Areas of application for immunological methods
In modern laboratory practice, immunological methods are used in three fundamentally different directions:
- Solving non-immunological tasks. Immunochemical analysis is widely used for the quantitative determination of hormones and other physiologically significant molecules in blood serum. In the past, radioimmunoassay was used for this purpose; today, it is ligand-binding assay (primarily ELISA) and immunoblotting. The advantages of these methods include extremely high specificity, sensitivity, and simplicity. However, their relative cost-effectiveness has been lost due to the high price of modern recording equipment. These methods belong to immunodiagnostics proper only when measuring molecules involved in immune processes (antibodies, cytokines).
- Assessment of immune status. This is a comprehensive characterization of the immune system's state without aiming to identify a specific immunopathology. It is used to study regional characteristics of immunity, assess environmental impacts, or link immunity to the HLA genotype. In clinical practice, there is a certain "detachment" of status assessment from real treatment tasks.
- Immunodiagnostics (in the narrow sense). Aimed at searching for specific abnormalities in a patient. The main ethical issue in immunodiagnostics is prescribing expensive tests that neither facilitate diagnosis nor contribute to improving therapy.
Method evolution and cell isolation
Historically, immunodiagnostics began with the diagnosis of infections, allergopathology, blood typing, and tissue compatibility. In the 1970s, a paradigm shift occurred: physicians began massively evaluating lymphocyte subpopulations and serum immunoglobulin concentrations. The goal shifted from diagnosing active infectious processes to identifying immunodeficiency states.
A key breakthrough in immunology was the development of a simple method for isolating lymphocytes (often called mononuclear cells due to the admixture of other cells).
- Principle: centrifugation of blood on a density gradient.
- Medium: a mixture of the polysaccharide Ficoll and a radiopaque agent (Verografin, Hypaque, or Omnipaque).
- Result: after centrifugation, a pellet forms at the bottom (erythrocytes, segmented neutrophils, eosinophils, basophils, and most monocytes). A ring forms above the dense medium layer, where lymphocytes gather with an admixture of dendritic cells and remaining monocytes.
Current standards and challenges in clinical practice
Previously, rosette formation was used to identify cells, a method now recognized as completely inadequate. The current stage of development was made possible by hybridoma technology and the creation of monoclonal antibodies. This made it possible to identify cells precisely by the binding of antibodies to their receptors.
Currently, the vast majority of clinical immunology tasks are solved using two highly standardized technologies:
- Enzyme-linked immunosorbent assay (ELISA) for humoral factors.
- Flow cytometry for studying cells.
At the same time, the problem of lacking clear standards for the functional characterization of immune cells remains unresolved.
It is also worth noting the difference in diagnostic approaches. In domestic practice, immune status assessment has begun to be prescribed excessively, on par with routine blood and urine tests, although physicians often lack the qualifications to adequately interpret these data. At the same time, according to strict WHO guidelines, cell immunophenotyping is indicated only for diagnosing three conditions: AIDS, primary immunodeficiencies, and lymphoproliferative disorders.
How to remember cell distribution during density gradient centrifugation: heavy cells with segmented nuclei (neutrophils, basophils) and dense erythrocytes break through the gradient and fall into the pellet. Light cells with a single round nucleus (lymphocytes, mononuclear cells) float on top, forming a ring.
Class and exam questions
How does immune status assessment differ from immunodiagnostics?
Immune status assessment is a general study of the system without searching for a specific disease (e.g., to assess environmental impact). Immunodiagnostics is aimed at identifying specific abnormalities to diagnose a patient or adjust their treatment.
Which cells remain in the "ring" after gradient centrifugation?
Mononuclear cells gather in the ring above the density gradient: lymphocytes, dendritic cells, and some monocytes. Erythrocytes and segmented cells go into the pellet.
Why shouldn't you prescribe an immune status test "just in case"?
This is an expensive study whose results in general practice have low informational value and often do not affect treatment tactics. According to WHO guidelines, detailed immunophenotyping is indicated only when AIDS, primary immunodeficiencies, or lymphoproliferative disorders are suspected.
What else to study on this topic
- Principles of enzyme-linked immunosorbent assay (ELISA) and immunoblotting
- Design and operating principles of the flow cytometer
- Hybridoma technology and monoclonal antibody synthesis
- Mechanisms of density gradient centrifugation (Ficoll-Verografin)
- The link between immunity and the HLA genotype
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