Immunophenotyping

In order to be able to characterise a cell in human blood in relation to its malignancy, it is possible to use monoclonal antibodies to separate it from healthy cells based on its surface features (cellular protein structures).

Each cell has special cellular protein structures, called antigens, on its surface. They can be by bound by monoclonal antibodies that are tagged to fluorescent dyes. They undergo an antigen-antibody reaction.

When the cells that are to be tested are put under a laser beam, light from fluorochromes is absorbed and released again. In immunophenotyping, both the light scattering properties of a cell as well as the fluorescence-marked antibodies are detected and analysed. Each individual cell can thus be assigned to a specific cell population.

Test material
In principle, it is possible to immunophenotype all cell-rich body fluids. However,

  • peripheral blood (at least 2 ml)
  • bone marrow (at least 2 ml)
  • cerebrospinal fluid (> 100 cells/µl; at least 2 ml)
  • pleural or ascites fluid (at least 50 ml)
  • bronchoalveolar lavage (BAL) fluid (at least 25 ml)

should be preferred for immunological testing.

Preanalytics

  • Peripheral blood or bone marrow should preferably be taken in syringes containing heparin (lithium heparin without gel). When using EDTA as an anticoagulant, samples must be quickly sent to the laboratory and prepared (within 12-24 hours). The test material (undiluted, anticoagulated) is transported and stored at room temperature (18-22 °C).
  • The pleural or ascites fluid and the aspirated BAL fluid should be taken very quickly to the laboratory in a transport vessel while refrigerated.

Indications
The process of typing pathological sub-populations mainly involves characterising the immunological phenotype of leukaemia blasts or lymphoma cells from the peripheral blood, bone marrow and pleural and ascites fluid for:

  • Acute myeloid leukaemia (AML)
  • Acute lymphatic leukaemia (ALL)
  • Blast crisis chronic myeloproliferative syndromes (MPS)
  • Leukaemic forms of non-Hodgkin’s lymphoma (NHL).

The immunophenotyping of BAL fluid permits differential diagnostics and the assessment of interstitial lung disease activity (e.g. sarcoidosis, idiopathic pulmonary fibrosis, eosinophilic pneumonia and various forms of alveolitis).

Immune status
Moreover, the „simple” immune status is also determined from the peripheral blood according to the principle of immunophenotyping. This involves a quantitative determination of the lymphocytes and their subgroups (B lymphocytes, CD4 and CD8 T lymphocytes, NK cells and activated T lymphocytes). This test can be used to diagnose and monitor therapy of congenital or acquired immune defects, in particular in the context of HIV.

In drug-induced immunocompromised patients, such as those receiving chemotherapy (exception code 32012), where there is a reduction in CD4+ T cells (< 200/µl), antibiotic prophylaxis with 160 mg of trimethoprim (3x/week) is recommended to prevent opportunistic infectious diseases, in particular Pneumocystis carinii infection.

Assessment
The explanatory power of immunophenotyping lies in the classification of the pathological cell population that defines the haematological disease. Further tests and the initiation of specific therapy for the respective disease will depend on the results. Immunophenotyping serves as a differential diagnostics aid for haematological disorders.


Literature
Sack, U., Tárnok A., Rothe, G.: Zelluläre Diagnostik. Grundlagen, Methoden und klinische Anwendungen der Durchflusszytometrie. Karger Verlag (2007)
Schmitz G., Rothe, G.: Durchflusszytometrie in der klinischen Zelldiagnostik.
Schattauer Verlag (1994)

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