Lymphocytes



How to Obtain Non-coagulating Blood
- Heparin (5-10 IU/ml): activates antithrombin III, blocks the action of thrombin
- EDTA: binds Ca2+
- Citrate: binds Ca2+

CD antigens
•Antigens expressed on leukocytes
•Abbreviation o Custer of Differentiation
•Each antigen is designed: CD+ a number (CD1- CD340..) •The antigens were designated after they
were recognized and characterized – no system exists.

Marker:      Cell Subpopulation
CD3 , CD4, CD8 T-lymfocytes
CD19, CD20, CD21 B-lymfocytes
CD16/CD56 NK cells
CD14/DR monocytes
HLA-DR; CD25; CD69 activation markers
The most important CD antigens of lymphocytes

Flow cytometer –FACS
Fluorescence-Activated Cell Scanner (Sorter)


Flow cytometer
IMG_0063


Flow cytometry
•
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Flow-Cytometry-Diagram2

lymfocytes
monocytes
granulocytes
granularity

CD3
CD4


CD3
CD8


CD3
CD8


Laboratory investigation of lymphocytes
•Enumeration of lymphocyte subsets by monoclonal antibodies against typical cell-surface markers -
CD3 (T-lymphocytes), CD4 (helper T-lymphocytes),
CD8 (cytotoxic T-lymphocytes),
CD19 or CD20 (B-lymphocytes), CD16 (NK cells).
•
•Evaluation of function -lymphocyte proliferation tests - determines response to various stimuli:
–specific (antigen, anti CD-3)
– non-specific -polyclonal mitogens: PHA (Phytohaemagglutinin), ConA (Concanavaline A), PWM
(Pokeweed mitogen)

Delayed-type of hypersenzitivity test



Tuberculin test









Cell-Mediated Immunity (CMI) tests
•Uses the principle of delayed type of hypersensitivity
•Common anamnestic antigens are used (tuberculin, candidin, trichofitin, staphylococcal antigens…)
•In normal situation induration shout be formed by 48 hours.
• If the patient does nor react to majority of antigens a deficiency of T- cell mediated immunity
is suspected.

Investigation of function of neutrophils
•Baktericidial killing of various bacteria of fungi
•Chemotaxis (e.g. under agarose)
•Ingestion of inert particles
•Metabolic activity - production of reactive metabolites of oxygen
–NBT test (reduction of colourless nitro-blue-tetrazolium into blue formasan)
–“Burst test“ – tetrarrhodamide reduction – measured by flow cytometer.

NBT