flowcytometry &cytolytics (4)

Presentation Possibilities of FACS-Data

Flow cytometry (Fluorescence-Activated Cell Sorting, FACS) is a method for the analysis and preparation of particles in mixtures of substances based on scattered light and fluorescence properties. The high analysis speed and sensitivity as well as the objective quantification and multiparametric correlations (relationship of at least two variables) open up an almost unlimited field of applications for flow cytometry in research and diagnostics. The focus of interest is always the individual cell. In contrast to traditional biochemical and cytochemical methods, no average values of a cell preparation are obtained, but the correlation of the result with the individual cell is maintained. Analysis techniques rely on representations using one-dimensional (e.g. histograms), two-dimensional (e.g. dot-plot) figures, and even higher-order graphs (plots) (3D-plots, SPADE trees, etc.).

Histogram

The most common and well-known evaluation representation of flow cytometry is the histogram. It represents a frequency distribution of the measured signals of a parameter. Typically, figures with data from different conditions are shown in one diagram. The horizontal axis represents the intensity of the individual measurements and the vertical axis represents the number of cells. In this way, the Gaussian distribution of a parameter is obtained, which is called the population. When cells are stained with fluorescently labeled antibodies, the fluorescence intensity is directly proportional to the number of binding sites (antigens) present, i.e., the more binding sites there are, the brighter the cell glows. Flow cytometry thus shows the distribution of different fluorescence intensities using a relative scale. The user must specify which areas should be considered “positive” or “negative” to properly evaluate a given population. These ranges are defined with control. Histograms are useful for cell cycle and proliferation analyses but are less useful for plotting data for several reasons. First, relationships between different markers will not be detected, i.e., double-positive cells cannot be identified. Second, small populations are lost in larger distributions, thus rare events are not noticed.

Dot-plot

If two different parameters are recorded during one measurement, a histogram is not sufficient and a two-dimensional representation is used. It allows to show the correlation distribution, i.e. the relationship between two different characteristics, and thus to identify more complex phenotypes. Thus, the populations in demand can be isolated using gating. The original two-dimensional plot is also known as a “dot plot,” a graph that showed the relationship between two traits but lacked detail in terms of the intensity of the number of events in a given region. Therefore, two-dimensional plots have some utility in showing how populations of interest are identified.

Scatter graphs

Another form of representation is a scatter graph, which can show information about dependency structures of two defined characteristics. The data are shown as scatter graphs, in which distributed characteristics can overlap if the same values are present several times. From scatter graphs, various focal points can be shown, such as the number of experiments performed to generate the data, or the mean, dispersion, and significance of the data.

Population

In flow cytometry, frequency distributions (populations) of cells are defined by their scattered light parameters – forward scattered light (FSC) and side scattered light (SSC) – and by their fluorescence. Since this is a relative measurement, controls must be used to define what is considered “positive” or “negative”. In the subsequent analysis, first, a pre-selection of the raw data is made (FSC versus SSC gate) and then the boundary between a negative and a positive population is defined, often based on a negative control (“threshold method”). A gate (a defined region) can of course also be set on the fluorescence parameters, for example, to select a specific lymphocyte population. All gates can be linked together. Thus, the gates act like filters connected in series. 

Compensation

Compensation is the proportional subtraction of a noticeable neighboring fluorescence in overlapping fluorescence spectra. In compensation, a relative amount is subtracted from a fluorescence signal by calculation and the difference in light quantity is referred to as the compensated signal. A disadvantage of compensation is that the positive population, due to the logarithmic amplification, is pulled apart in the direction of compensation, i.e. it is scattered more widely.

Gating

Gating is the process of defining a group of cells and gating them into another plot. However, since more than one cell population or property is studied in the research, gating can be very time-consuming. It is also a common criticism of flow cytometry data in general, as it represents a subjective evaluation.

With the proliferation of new automated analysis techniques, this problem is also being addressed while assuring that the data extracted for downstream statistical analysis comes from a robust, peer-reviewed process.

 

About the author
Elif Karakurt
medical content creator
Elif is a medical student and works for Cytolytics in the branches of content creation and marketing alongside her studies. She is the head of the Cytolytics blog and could already gather experience in writing medical articles for various magazines. Her interests are recent health issues and news about medicine, health technologies, and digital health.
flowcytometry &cytolytics (3)

Cancer

With approximately 230,000 deaths per year, cancer is the second most common cause of death in Germany (first place: cardiovascular disease). Due to the aging process, the number of new cancers occurring each year is increasing. Currently, the most common cancer for men is prostate cancer, followed by lung and colon cancer. For women, on the other hand, breast cancer is the most common cancer, followed by colorectal and lung cancer. This leaves most (affected) people wondering what cancer is and how it occurs. How cancer develops has not yet been clarified in detail. However, it is known that all types of cancer have in common a degenerate development of originally healthy body cells. They grow uncontrolled, into the surrounding tissue, and thus have a fatal functional restriction of the affected organs. These cancer cells can originate in almost any organ and migrate to other organs and lymph nodes via blood vessels or lymphatics. From there, they can develop into metastases, which also grow into healthy tissue just like the main tumor. In the case of cancer, therefore, survival depends on the timing of the cancer diagnosis, since cancer in its early stages has a good chance of cure and is usually not metastasized. Diagnostics play a key role in this: nowadays, cancer is diagnosed with the help of laboratory tests. To develop fast and targeted control examinations and therapy concepts, good laboratory equipment is, therefore, a mandatory requirement of every clinic. The faster and more accurately cancer is detected, the greater the chance of a successful cure. 

What actually is cancer?

 The body of every creature is made up of countless different cells, all of which have different tasks. An association of cells is called a tissue, which is a component of an organ. Each cell has a certain lifetime, therefore it can form new (and identical) cells by its division to maintain the organ function. If the body cells are healthy, their growth, development, and division as well as their death proceed without any problems. Cancer is a pathological change in the cells of the body. In this case, uncontrolled multiplication occurs due to rapid and defective division. So that they eventually form a lump (tumor). A tumor is defined as a hardening or swelling. They can be benign or malignant. The malignant tumor cells are often dangerous, as these cells can invade deeper and deeper into adjacent tissues and form metastases. The difference between benign and malignant tumors is no benign cells reach healthy tissue. Benign tumors include lipomas (fatty growths), moles, hemangiomas (vascular growths), and myomas (muscle cell growths).

How does cancer develop?

Taking a closer look at the development of cancer, there are various misregulations in the cell’s division. This disturbing process is called carcinogenesis, which originates in a cell whose genetic material is altered (by inheritance or independently acquired). This change (defect) is not reversed by misregulated repair mechanisms and is passed on accordingly during cell division. The older the human being gets, the more unreliable the repair system of the genetic material works. With further proliferation, these defective malignant cells can displace and damage the healthy cells. Among the best-known and most frequent causes, besides old age, are cigarette smoke, alcohol, sunlight, or radioactive radiation. Besides, there is a whole range of other causes of genetic changes, which would be too much to list here. However, it should be known that lifestyle such as diet, exercise, alcohol consumption, and smoking, in addition to external factors such as sunlight and pollutants, play a major role in the development of cancer. Accordingly, some of these risk factors can be influenced, while others cannot. It is estimated that about one-third of cancers could be prevented by avoiding risk factors such as smoking or alcohol. The remaining diseases are due to factors that cannot be influenced or are unknown.

What are the cancer classifications?

Cancer can be classified according to organ, cell of origin, characteristics of the tumor tissue (dignity), appearance type of the tumor (phenotype), diversity of cells within the tumor (grading), state of spread (staging), and metastasis of the main tumor. The classifications and tumor type are important for subsequent therapy and control examinations, as they allow more targeted planning and treatment. The internationally standardized classification is the TNM-classification (tumor, “nodus” or lymph nodes and metastases). These classifications are highly relevant for every type of cancer in every possible organ (from the brain to the urinary bladder) and vascular system. The classification has the advantage of standardizing cancer studies worldwide for research and prognostic purposes, thus enabling better treatment planning.

How can cancer be diagnosed?

Early diagnosis of cancer is extremely important because it significantly improves the prospects for treatment. Thus, the chances of recovery increase the earlier the cancer is detected. The first step in diagnosis is a detailed interview and physical examination of the patient. There are various examination methods for localizing the exact location of cancer. For example, blood tests, imaging procedures (ultrasound examination, X-ray, computer tomography, or MRI), and taking tissue samples (biopsy) provide important information about the organ, metastases, and corresponding functional impairment of the body. There are also so-called tumor markers in the blood, which are the body’s substances that proliferate in some cancers. These substances are either formed by the cancer cells themselves or are stimulated to form. These tumor markers are usually determined during control examinations after successful treatment to rule out a recurrence of cancer.

How can cancer be treated?

Since every cancer is different from person to person, it is essential to tailor treatment to the individual. Each patient has different requirements for certain therapies. Therefore, the general condition and concomitant diseases of the patient must be strictly considered beforehand. Thus, the optimal cancer treatment depends on the type of cancer as well as its spread. Treatment usually consists of three basic pillars: surgery, chemotherapy, and/or radiotherapy. In surgery, depending on the condition, cancer can be completely removed, chemotherapy stops the uncontrolled altered cell division by special cell toxins (cytostatics), and if necessary, radiation therapy can also be used to target cancer cells with electron radiation or X-rays. This procedure causes targeted damage to the cancer cells, which in turn causes cancer to decrease in size or stop growing. In addition to the three therapy options mentioned, other treatment options are used depending on the particular type of cancer, such as hormone therapy for hormone-dependent prostate or breast cancer, or antibody therapy (immune treatment), in the course of which special antibodies attack the cancer cells. When considering the therapy concept, a clear objective should also be defined, i.e. is the treatment completely curative, or does it only alleviate the symptoms and improve the quality of life for a certain period. Therefore, as a person affected or a close caregiver, one should not immediately think of a death sentence when cancer is diagnosed. If the serious disease is detected at an early stage, a cure is often possible. For this reason, it is advisable to attend cancer screening examinations regularly.

About the author
Elif Karakurt
medical content creator
Elif is a medical student and works for Cytolytics in the branches of content creation and marketing alongside her studies. She is the head of the Cytolytics blog and could already gather experience in writing medical articles for various magazines. Her interests are recent health issues and news about medicine, health technologies, and digital health.