STANDARDIZATION OF TISSUE SPECIMEN PRESERVATION BY ULTRASOUND AND TEMPERATURE CONTROL
A tissue sample preservation process and device for improving and standardizing tissue preservation procedure, including placing tissue specimens in a cold fixative, performing fixative penetration at a refrigerated temperature, and accelerating fixative penetration by ultrasound. Also disclosed is the application of ultrasound and temperature control in the dehydration, clearing, and impregnation steps.
1 . A method of preserving tissue specimens comprising:
a) submerging said tissue specimens into a fixative of a temperature between 0 to 25 degree C.,
b) subjecting said tissue specimens and said fixative to a cooling mechanism, and,
c) subjecting said tissue specimens and said fixative to a convection mechanism.
2 . The method of claim 1 , wherein said fixative is a formaldehyde water solution comprising 1% to 10% of formaldehyde by weight, and wherein said temperature is between 4 to 20 degree C., preferably between 4 to 18 degree C.
3 . The method of claim 1 , wherein said fixative is a solution comprising at least 50% by volume of at least one chemical from the following group: ethanol, methanol, PEG, and acetone; and
wherein said temperature is between 4 to 25 degree C., preferably between 4 to 18 degree C.
4 . The method of claim 1 , wherein said convection mechanism is ultrasound irradiation.
5 . The method of claim 4 , wherein said ultrasound is of a frequency between 0.1 MHz to 5 MHz, preferably between 0.2 MHz to 2 MHz, and further preferably between 0.4 MHz to 1.5 MHz.
6 . The method of claim 5 , wherein said ultrasound is of a power between 1 to 100 Watts, preferably between 5 to 50 Watts, and further preferably between 10 to 30 Watts.
7 . A method of claim 6 further comprising at least one dehydration step of submerging fixed tissue specimens in a dehydration solution of a temperature between 0-25 degree C.
8 . A method of claim 6 , further comprising: d) turning off said cooling mechanism after a predetermined length of time; e) continuing ultrasound irradiation to increase temperature of said tissue specimens in said fixative to a predetermined value; f) switching ultrasound irradiation on and off to maintain said predetermined temperature value for a predetermined length of time.
9 . A method of claim 6 further comprising:
at least one dehydration step; comprising: i) submerging fixed tissue specimen in a dehydration solution of a temperature between 0-40 degree C., ii) raising the temperature of said dehydration solution to a predetermined value by ultrasound irradiation alone or in combination with other heating means, and, iii) keeping the temperature value for a predetermined length of time;
at least one clearing step, comprising: i) submerging dehydrated tissue specimen in a clearing solution, ii) raising the temperature of said clearing solution to a predetermined value by ultrasound irradiation alone or in combination with other heating means, and, iii) keeping the temperature for a predetermined length of time:
an impregnating step, comprising: i) submerging the dehydrated and cleared tissue specimen in melted wax, ii) irradiating said dehydrated and cleared tissue specimen in melted wax with ultrasound for a predetermined length of time, and, iv) applying a vacuum to said dehydrated and cleared tissue specimen in melted wax.
10 . The method of claim 1 , wherein said convection mechanism is by stirring, agitation, or pumping-in and pumping-out.
11 . The method of claim 1 , further comprising:
d) subjecting said tissue specimens and said fixative to low pressure, wherein said low pressure is in the range of 10 to 20 inch Hg.
12 . The method of claim 1 , further comprising:
d) subjecting said tissue specimens and said fixative to high pressure, wherein said high pressure is a pressure that is above atmospheric air pressure.
13 . The method of claim 1 , further comprising:
d) subjecting said tissue specimens and said fixative to alternative high pressure and low pressure.
14 . A device for preserving tissue specimens in an ultrasound field, comprising:
1) At least one vessel for holding tissue specimens and preservation reagent(s),
2) An ultrasound generating system to produce ultrasound field in reagent(s), and
3) A temperature control system for maintaining a preset temperature in said vessel.
15 . A device of claim 14 , wherein said ultrasound generating system produces ultrasound signal of:
1) a frequency in the range of 0.1 MHz and 5 MHz, preferably in the range of 0.2 MHz and 2 MHz, further preferably in the range of 0.4 MHz and 1.5 MHz; and, 2) a power in the range of 0.1 to 100 Watts, preferably between 5 to 50 Watts, further preferably between 10 to 30 Watts.
16 . A device of claim 15 , wherein said ultrasound generating system comprises at least one piece of piezoelectric transducer, said piezoelectric transducer can be in the shape of a circular disc, a rectangle bar, a cylinder, or an array of multiple transducers.
17 . A device of claim 16 , wherein said piezoelectric transducer(s) is attached to the surface of the wall or the bottom of said vessel by a glue. 26
18 . A device of claim 16 , wherein said piezoelectric transducer (s) is sealed in a transducer housing.
19 . A device of claim 18 , wherein said transducer housing is submerged in said tissue preservation reagent(s).
20 . A device of claim 18 , wherein ultrasound is produced from one side of said transducer housing to irradiate on tissue specimens that are placed on the same side of said transducer housing emitting ultrasound.
21 . A device of claim 18 , wherein ultrasound is produced from both sides of said transducer housing to irradiate on tissue specimens that are placed on both sides of said transducer housing.
22 . A device of claim 15 , wherein said ultrasound generating system can deliver continuous or pulsed ultrasound field with fixed or sweep frequency(s).
23 . A device of claim 14 , wherein said vessel contains a side wall, a detachable cover, and a movable bottom fitted water tight to the side wall; said movable bottom is connected with a revolving engine; said revolving engine drives back and forth movements of said moveable bottom.
24 . A device of claim 14 , wherein said vessel contains a side wall, a bottom, and a movable cover;
said movable cover is fitted water tight to the side wall and is connected with a revolving engine; said revolving engine drives back and forth movements of said movable cover.
25 . A device of claim 14 , wherein said temperature control system contains a cooling means and a heating means.
26 . A device of claim 24 , wherein said cooling means is circulating a cold liquid through a space surrounding said vessel; said space is formed between the outside surface of said vessel and a shell surrounding said outside surface of said vessel; and said cold liquid is of a temperature between −20 degree to 25 degree C.
27 . A device of claim 24 , wherein said cooling means is circulating a cold liquid through a pipe passing through said tissue preservation reagent.
28 . A device of claim 24 , wherein said cooling means is a semiconductor device.
29 . A device of claim 24 , wherein said heating means is ultrasound irradiation alone or in combination of ohmic heating or microwave heating.