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Cytotoxicity Test Neutralization Measurement of Cell Mediated Immune Response
Cytotoxicity Test Neutralization Measurement of Cell Mediated Immune Response
Cytotoxicity Test Neutralization Measurement of Cell Mediated Immune Response
CYTOTOXICITY TEST
- Secondary binding test (like Precipitation, Agglutination and Complement Fixation)
- Detect cell destruction as a result of Ag-Ab reaction in presence of complement
- Complement may cause damage to the cell
- If there is no Ag-Ab reaction the complement cannot fix to the cell. Thus the three (Ag,
Ab and Complement) must be present in the test.
- Dye is added- only damaged cells will take up the dye
(Trypan Blue and Eosin Y)
Uses
HLA typing
Transplant surgery
Mediated Application
1. In transplant surgery
- Detects the closest match for transplant
2. Identification of HLA antigen associated with diseases
HLA DR4 Rheumatoid Arthritis
HLA B8 Graves disease (goiter), Hashimoto Thyroiditis (autoimmune disease that
starts as hyperthyroidism and after a time present as hypothyroidism), Sarcoidosis
(enlarged lymph nodes)
HLA B27 Ankylosing Spondilitis arthiritis in the backbone ( 85% of px infected with
A.S. Presents with HLA B27), Graves disease, Reiters disease ( present with
symptoms of arthritis, conjunctivitis and urethritis)
A13-B17 Psoriasis
NEUTRALIZATION TEST
- Secondary binding test
- Ag-Ab reaction in which specific antiserum is able to diminish/abolish some biologic
property of Ag but not its antigenicity:
Toxicity
Infectivity
Enzymatic activity
- An important mechanism in host defense against
o Extracellular viruses
o Most infective bacteria
o Microbial toxins
Antitoxin
- specific toxin neutralizing Ab
- Protective Ab that inactivates reaction produced by microorganism
Uses
1) To identify its corresponding toxin
2) To identify certain viruses isolated from patients (w/ a stock of known virus Abs in
patients serum can be Identified)
Toxoids
- Toxin treated with formaldehyde (process of attenuation) to make toxin harmless while
maintaining its antigenic activity
Uses
1) Immunization to induce formation of antitoxins (e.g. DPT)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
1:10 1:100 1:500
Serum 0.8 0.2 1 0.8 0.6 0.4 0.3 1 0.8 0.6 0.4 0.2 0.0 0.0
B saline 0.2 0.8 0 0.2 0.4 0.6 0.7 0 0.2 0.4 0.6 0.8 1.5 1.0
Shake tube gently + mix
Strep O 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.0 0.5
RGT
Shake gently, incubate at 37C for 15 mins
O cells 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Shake gently, incubate at 37c for 45 mins
Todd 12 50 100 125 166 250 333 500 625 833 1250 2500
Units
A titer of 166 IU/mL or 166 Todd Units is used since 200 is not part of the serial dilution
Tube 13: (RBC Control) : should show no hemolysis (+)
Tube 14: (SLO Control) : should produce complete hemolysis (-)
- Most persons respond with delayed type reactions to skin test Ag because
of past exposures to these Antigens
- Delayed type Hypersensitivity is mediated by T CELLS, not Ab
- It is dependent upon the release of lymphokines from activated T cells that
attract & hold inflammatory mononuclear cells at the skin test area
- Macrophage and Monocyte Chemotactic Factors lymphokine
- Main cells involved are: CD4 (helper) T cell and Macrophages
Procedure:
1) Intradermal injection of Ag into a suspected sensitized host
Ag will be recognized by CD4T helper cells which will secret the lymphokine:
MMCF Macrophage & Monocyte Chemotactic Factor, which when release
attracts monocyte and macrophage
2) A reaction at site of injury occurs as a red bump/lesion (w/in 24-48 hrs 72 hrs
max)
Lesion: firm, indurated (hard), non-edematous, erythema, red swelling
Histology: shows mononuclear cells, basophils and few neutrophil
*** if it is Ab mediated: shows neutrophil infiltration and edema
- Same procedure with the Tuberculin test but differs in the Ag used
- Used to diagnose Coccidioidomycosis
- Ag used: Mycelial extract of Spherule
- (+) test = induration on skin is produced by patients with Coccidioidomycosis,
which indicates past or present infection with C. immitis
- When some Ag is applied to same area 1-2 weeks later, they respond with a
delayed type skin reaction
- Immunocompromised patient with immunocompetent CMI produce a (-)
result
PROCEDURE
1) High dosage of DNCB is directly applied to skin (volar aspect of forearm)
2) 1-2 weeks later, a lesser dose is applied to the same area
(+) result = FLARE (erythema of skin that spreads outward), vesicular reaction,
itching, eczema, necrosis within 12 48 hours
C) GRAFT REJECTION
- Done only in animal study
- Transplantation of Organ tissue between non -identical strains of mice
result (+) to Graft rejection
Example is the use of Skin Grafts
- If a skin graft from an unrelated donor is applied, it is recognized as non-self
and rejected by the recipient within 1-2 weeks
- The transplant is attacked by T cells that directly lyse the grafted cells and by
macrophage activated by T cells
- The main immune cells involved are CD8 Cytotoxic T cells) and macrophages
cells
PROCEDURE
1) Add suspension of purified lymphocyte (by Ficoll Hypaque)
2) Mix with Mitogen
3) Cultivate for 48-96 hours
4) 12 hours before end of incubation period (cells are actively dividing)
5) Add the indicator, 3H Thymidine, a radiolabeled nucleotide (only actively dividing cells
take up stain)
6) Harvest (using automated harvester) and wash cells
7) Transfer to a tube containing scintillating fluid
8) Measure radioactivity of 3H Thymidine as CPM (counts per minute) using Scintillation
Spectrophotometer
- Non dividing cells do not take up Thymidine, while dividing cells take up
thymidine which means that they are actively synthesizing DNA
- Number of myeloblast formed is proportional to uptake of thymidine &
functional competence of lymphocyte which determines immune
responsiveness of patient
- Normal control must be run simultaneously with the patient lymphocyte in
lab
- To obtain the simulation index: =
>1 = normal <1 = not normal
Retyped by : Khayla Rondobio
B) Mixed Lymphocyte Culture / MIC
PROCEDURE
lymphocyte from 2 individuals when mixed together stimulate each other to undergo
blast transformation and proliferate
Proliferation
In Bone Marrow transplant, the proliferation of the donor cells in response to the
recipient cells indicate poor prognosis, an example of Graft vs Host.
When to do BM transplant
a) Severe Combines Immunodeficiency disease (SCID)
b) Aplastic anemia
c) Leukemia