Molecular-genetic approaches
Nucleic acid amplification technologies occupy an important place in the arsenal of modern laboratory diagnostics, allowing the study of the immune system at the gene level.
- Qualitative polymerase chain reaction (PCR) is actively used for genetic typing. In particular, this method is indispensable for determining HLA system alleles, especially when it comes to class II antigens.
- Quantitative real-time reverse transcription PCR is a promising tool that is being actively introduced into routine practice. With its help, specialists can accurately measure the expression level of specific genes encoding immunologically significant molecules.
Specifics of evaluating immune cell function
Unlike strictly regulated biochemical assays, methods for assessing the functional activity of immune cells are characterized by colossal methodological diversity. Because of this, they are significantly less standardized compared to other laboratory technologies.
Approaches to analyzing innate and adaptive immunity mechanisms have substantial differences. Thus, for assessing the state of innate immunity, routine counting of the quantitative composition of myeloid cells (via a standard complete blood count) has little diagnostic value in the context of their functional capability. Therefore, the main emphasis here is placed almost exclusively on functional tests.
Study of phagocytic activity
Phagocytosis is a basic defense process provided by innate immunity. Two main approaches are used to study it:
- Traditional (microscopic) method. The physician examines stained smears to which objects for uptake (microorganisms or inert latex particles) have been preliminarily added. Two key indicators are calculated:
- Phagocytic index: the percentage of cells that successfully captured the test particles.
- Phagocytic number: the average number of particles engulfed by a single active cell.
Disadvantages of the classical approach: high labor intensity, influence of the researcher's subjective opinion, and extreme difficulty in standardizing the results.
- Modern method. Based on flow cytometry. Instead of visual counting, bacteria or particles preliminarily labeled with fluorochromes are used, allowing the device to automatically register the engulfment activity.
Analysis of bactericidal and secretory function
The ability of a phagocyte to capture a pathogen is important, but from a functional perspective, it is much more significant to assess bactericidal activity—the ability to digest and destroy the target.
- Phagocytosis completion test. The classical version is based on assessing how much the number of viable microorganisms cultured from phagocyte lysates decreases. Today, simpler flow cytometry-based analogs of this test have also been developed.
- Nitroblue tetrazolium (NBT) reduction test. A method for indirect assessment of bactericidal potential. Cell activity is determined by the visual appearance of blue staining—the result of intracellular deposition of formazan granules.
- Assessment of reactive oxygen species (ROS) generation. An advanced technique based on the recording of chemiluminescence. Luminophores (lucigenin or luminol) are added to the sample, and a specialized device (chemiluminometer) records the emission. Registration occurs automatically, evaluating both spontaneous and stimulant-induced reactions.
Another important parameter of innate immunity is secretory activity. During the in vitro test, the ability of cells to produce pro-inflammatory cytokines (IL-1β, TNFα, etc.) is studied. Blood leukocytes or isolated macrophages are stimulated with bacterial agents—most commonly lipopolysaccharide (LPS) from Escherichia coli—after which the level of cytokine release is measured.
Briefly: answers to topical questions
Which pro-inflammatory cytokines do cells produce when assessing secretory activity?
When assessing secretory activity, innate immunity cells produce pro-inflammatory cytokines, primarily IL-1β and TNFα. In general, activated monocytes and macrophages are capable of producing a wide range of these substances:
- Tumor necrosis factor alpha (TNFα) is a member of the main pro-inflammatory cytokine triad.
- Interleukin-1 (IL-1, including IL-1β) is a member of the main triad.
- Interleukin-6 (IL-6) is a member of the main triad.
- Interleukin-12 (IL-12) is produced by macrophages.
- Interleukin-23 (IL-23) is produced by macrophages.
- Interleukin-27 (IL-27) is produced by macrophages.
- Interleukin-17 (IL-17) is an additional cytokine.
- Interleukin-18 (IL-18) is an additional cytokine.
Lipopolysaccharide (LPS from Escherichia coli) is most commonly used to stimulate cytokine production in vitro.
Which phagocytic enzyme system is responsible for the nitroblue tetrazolium reduction reaction?
The provided materials do not name the specific enzyme system directly responsible for the nitroblue tetrazolium reduction reaction. It is only indicated that the NBT test serves as a method for indirectly assessing the bactericidal potential of phagocytes, during which formazan granules are deposited inside the cell. At the same time, the main event of bactericidal activity is the respiratory burst, and the key enzyme catalyzing the formation of reactive oxygen species is NADPH oxidase (phagocyte oxidase).
To avoid confusing phagocytosis indicators: INDEX is a PERCENTAGE (the proportion of cells that started working), while NUMBER is a COUNT (how many particles a single cell ate).
Class and exam questions
Why are functional tests specifically used for innate immunity?
The quantitative composition of myeloid cells is easily determined using a routine complete blood count. However, the count alone does not reflect the cells' actual ability to protect the body, which is why tests evaluating their function are necessary.
What is the fundamental difference between the phagocytic index and the phagocytic number?
The index shows the percentage of cells that were able to perform phagocytosis at all. The number reflects the individual "capacity"—the average number of particles engulfed by a single cell.
What acts as an inducer in assessing macrophage secretory activity?
In laboratory practice, cells are stimulated in vitro with bacterial agents. Lipopolysaccharide (LPS) derived from Escherichia coli is most commonly used to trigger the production of pro-inflammatory cytokines.
What is qualitative PCR used for in clinical immunology?
This molecular-genetic method is most frequently used for precise typing of major histocompatibility complex (HLA) antigens, especially class II molecules.
What else to study on this topic
- Mechanisms of reactive oxygen species generation by phagocytes
- Principles of flow cytometry in immunology
- The role of pro-inflammatory cytokines IL-1β and TNFα in the immune response
- Quantitative real-time reverse transcription PCR
- Structure and functions of major histocompatibility complex (HLA) molecules
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