What is an LTT Assay?
A Lymphocyte Transformation Test (LTT) is also called a lymphocyte proliferation test. This test measures the amount of reactivity (or proliferation) of immune cells called lymphocytes, after they have been exposed to a particular challenge (such as a type of metal or a kind of drug). Lymphocytes are central cells of our immune systems that react to a threat in several ways.  One way they do this is to expand in number. People with a sensitivity to a particular metal will have lymphocytes that think the metal is a threat and will mount a defense.  This causes inflammation.

How is the metal-LTT test conducted?
1. A blood draw is necessary to collect the lymphocytes and other important immune cells (like monocytes).
2. The important types of immune cells (PBMCs) are isolated from the blood and collected.
3. This collected fraction of immune cells (containing lymphocytes) is divided up in a culture dish and each part is exposed to a different metal. 
4. These
lympohcytes are cultured for a little less than 1 week in the presence of metals that are in different types of orthopedic implant metals (including Aluminum, Cobalt, Chromium, Iron, Molybdenum, Nickel, Vanadium and Zirconium).
5. After the cells have been grown together with the metals for 5-6 days the amount of proliferation responses the lymphocytes had is measured.
 6. The results are analyzed and sent out.

Why use metal-LTT?
The clinical observations of unexplained pain, effusion, stiffness and/or cutaneous eruptions following total joint arthroplasty have increased the need for a more accurate methodology for diagnosing "metal allergy" (hypersensitivity) to metallic biomaterials.
Current methods used to diagnose hypersensitivity reactions, such as dermal patch testing, are not well accepted in orthopedic practice, and involve the possibility of inducing hypersensitivity responses. To address this need we have developed an in-vitro LTT assay to test for metal sensitivity to Aluminum, Chromium, Cobalt, Iron, Molybdenum, Nickel, Vanadium, and Zirconium.

This assay facilitates a dose response quantification of metal-induced hypersensitivity responses in terms of generalized lymphocyte reactivity (i.e. proliferation). Issues of sensitivity and specificity remain unresolved as well as how implant performance is related to positive reactivity results. Metal-specific reactivity is gauged by comparing non-treated to treated lymphocytes from the same individual and categorized using the following general criteria: 2-4 fold response =mild reactivity, 5-8 fold =moderate reactivity, and >8 =high reactivity.

Technical Protocol: Proliferation Assay (Lymphocyte Transformation Tests): Proliferation of cells is measured by [3H]-thymidine (Amersham International, Arlington Heights, IL) incorporation into DNA in a 96-well microplate system. The average for each treatment is normalized to that of the negative control (no treatment) producing a ratio, generally termed a proliferation factor, proliferation index, proliferation ratio or stimulation index, SI. The SI is used to compare lymphocyte reactivity to the different metals. The lower limit of this stimulation index is zero indicating all cells stopped dividing before addition of [3H]-thymidine, after 5 days. Proliferation assays are performed using Ficoll separated peripheral blood mononuclear cells (PBMCs) collected from 30-40 milliliters of peripheral blood. These isolated PBMCs are cultured in 96-well cell-culture plates (Sigma), at a density of 0.1-0.3x10^6 cells/well for a period of 6 days in 150 m L of DMEM/well, 10% autologous serum at 37degrees C and 0.5% CO2, with metal treatments, a positive control (0.01 mg/ml PHA) and a negative control (untreated). Each treatment is conducted in triplicate (3 wells/treatment). [3H]-thymidine is added during the last 12 hours of incubation after 5 days of treatment. At day six [3H]-thymidine uptake (1 m Ci/culture well) is measured using liquid scintillation. The SI is calculated using measured radiation counts per minute (cpm): Simulation Index = (mean cpm with treatment) / (mean cpm without treatment).

Six days of incubation are chosen to reproduce, in vitro, the time lag associated with in vivo lymphocyte proliferation in a DTH response. Radiolabeled lymphocytes are collected onto membranes using a cell harvester (Tomtech Mach 2, Orange, CT) and the amount of differential radiation incorporation is measured using liquid scintillation (Wallac 1205 Betaplate, Gathersburg, MD). Stimulation indices of 2-4 indicate mild reactivity, 5-8 moderate reactivity and above 8 high reactivity to metals .




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