Immunophenotyping, the process of analyzing the proportions of different cell types within a heterogeneous population, is most commonly performed by multicolor flow cytometry. Yet while the ability to identify cells using a hierarchical combination of antibodies is a critically important tool within both basic and clinical research, it can be extremely challenging and is becoming more so as immunophenotyping panels continue to increase in size and complexity. The growing diversity of markers employed for immunophenotyping has led to the evolution of multiple approaches designed to overcome the various difficulties that these present, and a range of enabling products and technologies have been developed allowing researchers to gain greater insight into cellular populations than ever before.
Mike Blundell, product manager at Bio-Rad, explains that immunophenotyping increases our understanding of a wide range of processes. These include immune responses to disease, malignancy, vaccine efficacy, clinical diagnosis, immune infiltration in cancer, and residual disease after treatment. “Not only does profiling indicate which cell types are present or responding, but it allows identification of new cell types and delivers information regarding the activation state, cell cycle, cell health, intercellular interactions, and homing ability” he says. “In fact, it is now possible to assess the efficacy of novel treatments through necrosis and apoptosis levels of specific subsets of cells, as well as determine which type of programmed cell death is occurring.”
“From a research perspective, being able to identify and sort specific sub-populations allows the study of the biological functions of these cells,” notes Alan Stall, principal scientist and BD Fellow, R&D at BD Biosciences. “Clinical applications include the identification of cells involved in auto-immune disease, and measurement of the relative proportions of different cell sub-populations as an indication and/or prediction of the overall health of an individual. The best-known example of the latter is exemplified by HIV infections, where the ratio of CD4+ to CD8+ T cells illustrates disease progression. Another instance is in leukemias and lymphomas, which are due to abnormal expansion of a single type of leukocyte; the phenotype of the expanded population defines the type of leukemia or lymphoma and helps determine a treatment modality.”
Numerous markers can be used to identify cell type, those most commonly exploited for immunophenotyping being CD molecules. “These are mainly found on the cell surface, making their immunodetection straightforward,” says Blundell, “however the combination of surface and intracellular staining can be more problematic. As the desire to include chemokine receptors, cytokines, adhesion molecules, transcription factors, signaling molecules, and glycoproteins within immunophenotyping panels grows—with the aim of identifying further cellular subsets—lengthy optimization may be required due to antigen availability after fixation and permeabilization.”
Tips for success
Common tactics to increase the likelihood of successful immunophenotypic staining include using brighter fluorophores for low abundance targets, incorporating tandem dyes into staining protocols to maximize the number of readouts, and performing Fc blocking if cells are known to be Fc receptor positive. “Cell types that have a low frequency and low expression level can be especially difficult to identify,” reports Blundell “while cells from samples that require a lot of processing are more likely to have poor health leading to increased non-specific staining. The former can be overcome by using a bright fluorophore in combination with the acquisition of many cells, while the inclusion of viability dyes, dump channels to remove unwanted cells, and optimized staining protocols are essential to the latter.”
To avoid cross-linking and subsequent cell activation, many researchers are turning to antibody fragments such as Fabs or single domain antibodies. These lack an Fc region, making them considerably smaller than conventional IgG antibodies and affording the potential for binding to epitopes that may be inaccessible to larger antibody molecules. According to David Eling, director of business development at Pro-Sci, “finding antibodies with specificity and high affinity remains a challenge to those wishing to immunophenotype cells. We offer a broad range of antibodies for immunophenotyping, including a portfolio of highly specific llama single domain antibodies that are immune-reactive to multiple oncology targets”. In addition to their small size, further advantages of single domain antibodies are high solubility, and exceptional stability under extreme temperature and pH.
Standardized protocols
A growing area of concern within the field of immunophenotypic analysis is the need for protocols to be standardized. This was highlighted within a recent Nature publication discussing how, within a research setting, each study tends to use its own combination of markers and fluorochromes, even when purportedly analyzing similar cell subsets. One company very aware of the significance of this issue is Miltenyi Biotec, which is using an automated staining protocol and recombinant antibodies to improve the standardization of whole blood immunophenotyping assays. “Such screening can often take place within multicenter studies for long-term evaluation of patient cohorts, and it is therefore vitally important to reduce any human error,” elaborates Johannes Fleischer, global product manager.
“Two essential steps in the flow cytometric analysis of human whole blood are the lysis of erythrocytes and the staining of cell type specific markers,” he adds. “These are influenced by multiple parameters such as incubation time, temperature, sample handling, and choice of reagents, all of which can introduce significant variability. Automation of lysis and staining not only provides greater reproducibility, but it also decreases hands-on time.” The company recently presented a poster at Cyto 2018, comparing an automated lysis and staining protocol performed with their MACSQuant® Analyzer 10 to a manually performed experiment. “Automation resulted in faster lysis, with the detected erythrocyte population showing just one specific side-scatter intensity, whereas the manual procedure sometimes resulted in incomplete lysis, and two erythrocyte populations with different side-scatter intensities were detected. This provides a clear indication that switching to automated procedures is to be recommended.”
Image: Immunophenotyping of cells from samples lysed and stained via a manual or an automated process delivers similar cell counts, however the automated process provides greater reproducibility. The flow cytometry data exemplifies the results for an automatically processed sample. Image courtesy of Miltenyi Biotec.
Within this same comparative analysis, the researchers at Miltenyi Biotec also demonstrated that reproducibility could be improved by using the company’s recombinant REAfinity™ antibodies in place of hybridoma-derived equivalents. “These afford higher lot-to-lot consistency than traditional antibodies,” says Fleischer, “and since they are mutated at the Fc region to abolish any background binding to FcγRs, tedious and costly Fc receptor blocking steps can be eliminated from the staining protocol.”
According to Stall, the number of reagents available to the research community for immunophenotyping is increasing daily. “We used to consider NKT cells a singular population, yet over 25 sub-populations have now been identified,” he says. “As new markers are identified, the limitation in many cases is the ability to make high-quality binding reagents to detect these. As one means of addressing this, researchers are exploring new technologies to generate alternative binding molecules, for example aptamers, for immunophenotyping applications.”
Continued advancements in immunophenotyping reagents and technologies are crucial, Blundell explains. “Immunophenotyping allows real-time tracking and efficacy of novel immunotherapies, such as CAR-T cell therapy, to be assessed. It is also useful in evaluating iPS cell differentiation and manipulation and therefore suitable for potential therapies. Yet another expanding area of research is the immunophenotyping of different sample types like exosomes. Immunophenotypic profiling will be of huge importance to the future of precision medicine where specific treatments can be tailored to the individual.”