GB2337588A

GB2337588A - Retrieval of biosamples

Info

Publication number: GB2337588A
Application number: GB9911103A
Authority: GB
Inventors: Graham James Shorten
Original assignee: Individual
Current assignee: Individual
Priority date: 1998-05-16
Filing date: 1999-05-13
Publication date: 1999-11-24
Also published as: GB9911103D0; GB9810523D0

Abstract

A method of retrieving samples of biological material enables retrieval of an individual sample from a frozen array of such samples e.g. in the wells of a microtitre tray. Energy from a laser source of energy is applied to a single storage site within the array. The temperature of said site is monitored and, in dependence thereon, the power input to the laser source or the duration of input is adjusted to melt just the desired sample, leaving those samples surrounding it still frozen. Once the thawed sample is removed from a well, the remaining samples are returned to cold storage without having been disturbed.

Description

2337588 1 RETRIEVAL OF AN INDIVIDUAL SAMPLE OF BIOLOGICAL MATERIAL The

present invention relates to a method and apparatus for retrieval of an individual sample of biological material from a frozen assembly of such samples. More particularly, the invention relates to a method which enables such retrieval to be effected with less risk of contamination or damage to adjacent samples within the frozen assembly.

It is known to freeze samples of biological material such as blood or DNA containing 0 material and such samples may be kept for long periods often up to several years - at temperatures in the region of minus SO'C. At some time during the storage period, it may be required to access and retrieve a particular sample. However, it is usual to store samples in a microtitre plate having a large number of individual wells, often 96, 384 or more wells, each containing a sample of volume between 0.2mi to 2m1. In each case, the shape of the well will depend upon the manufacturer of the microtitre plate, but some have flat bases, some round 2 bottoms and some are conical or V-shaped wells. The well shape may either be round or square.

Retrieval of an individual sample from a multiple well microtitre plate as described, is presently carried out by raising the temperature of the entire plate to between 4' and 6'C, at which temperature an individual sample can be retrieved, following which the entire plate must be refrozen, thereby possibly degrading the remaining biological samples. The method of raising and lowering the temperature must be controlled so as not to rupture intercellular walls of the laboratory samples.

Such a method of retrieval has the obvious disadvantage of degrading the quality of the samples in the plate each time it is thawed to retrieve a sample and refrozen afterwards. There is also the danger of contamination between samples on the same plate while it is in the thawed condition.

In an attempt to overcome the above disadvantage, it has been proposed to retrieve samples by inserting a heated probe into a single well of the tray to thaw and then withdraw the sample. However, in order to avoid contamination, such a probe can only be used once without sterilization. Furthermore, great care is required since insertion of the probe may cause the sample to overflow and contaminate other wells in the same plate and, finally, the difficulty in controlling the rate of temperature rise of the sample makes the method very slow.

It is an object of the present invention to provide a method of recovery of individual samples which overcomes the above disadvantages.

1 i 3 According to the present invention, there is provided a method of retrieving an individual sample of biological material from a frozen assembly of such samples, said method comprising the steps of applying a laser source of energy to a single storage site within the assembly, monitoring the temperature of said site and, in dependence thereon, adjusting the power input to the laser source and/or the duration of input.

Use of a laser source to impart heat to the sample is controllable immediately with no residual temperature gain after the laser beam is switched off. If the sample is required to be maintained at a given temperature, the laser beam may be pulsed to ensure that the sample remains at the required temperature. The laser may be sited below, above or to one side of the sample tray. Preferably, the laser beam is directed to the underside of the sample tray, optionally via a reflective surface disposed movably beneath the tray.

The temperature of a particular sample may be monitored either by laser beam feedback or by checking infrared radiation emitted by the sample, and it is usual to stabilise the temperature of the sample at +4' to +6'C for retrieval thereof It is preferred that the power to the laser is pulsed to raise temperature gradually or maintain a temperature approaching the desired temperature.

Once the sample has been removed from its well, the plate containing the remaining samples may be replaced Into cold storage without the remaining wells of the sample having been disturbed.

4 The laser beam may be directed towards a particular sample by relative movement between the laser beam, i.e. the laser projector and/or a reflector and the sample tray along XY coordinates using stepper motors to move either the laser beam or the tray. The tray may be delivered to the retrieval zone either by a roller conveyor or by means of a robot arm to an identifiable point within the XY coordinates.

The entire retrieval process may be controlled using a microprocessor which controls the motors to align the laser beam and/or the sample tray according to the XY coordinates of the well, the contents of which are to be retrieved. The coordinates of the well may be indicated either by a keyboard, thumbwheel switches or keyboard controls or by barcode. The microprocessor may also control the laser intensity and monitor the sample temperature to give an output signal when the sample is retrievable. It may then enable retrieval of the sample and allow progress of the sample tray to further storage.

Initial tests were made on the various types of trays used for sample storage to determine transmission and/or absorption of energy at 8 1 Orim.

The tests were performed using a Coherent Diode laser with an output wavelength of 81Orim and a maximum energy of 9W CW Type FAP-808-12c-800-B.

Each test consisted of allowing the focal point of the laser beam to strike the underside of an individual sample cell of each tray for one minute. The focused beam diameter was IMM. The trays were then examined for possible heat damage, Tests were then made to confirm that a transfer of energy would be made to a sample within a cell. The laser beam was directed vertically upwards into each cell. The focal point was set to occur within the sample 3mm from the cell base. The sample liquid was composed of a 1: 1 solution of E 122 (red) food colour with water. Temperature readings were taken with a type K thermocouple and digital readout.

In each case, the Diode drive current was 1 SA; Irradiation time was 60 seconds- and the Sample volume was 220RI.

Nine types of tray were tested as follows:

Manufacturer Cell Base Material Temperature Rise 1 Polyfiltronics Cone Polypropylene, natural 18.70C 2 Unknown Flat Polystyrene 7.20C 3 Microtitre Cone Polystyrene 8.60C 4 Ritter riplate Cone Polystyrene 1 1.20C Unknown Cone Polypropylene 10.00C 6 Biometra Flat Polypropylene 8.OOC 7 Biometra Cone Polypropylene 11.40C 8 Greiner Cone Polypropylene, green No energy transfer 9 Unknown Flat Polystyrene, 384 cells 5.40C The results obtained from these tests indicate that energy transfer through the majority of tray types tested is good, the exception being the type 8 tray, in which the material of the tray I- 6 included green pigment. Improved heating is obtained on those trays with cone shaped sample cells, this is possibly due to reduced reflection of the laser beam entering the cell at an angle.

Individual natural polypropylene vials containing 100il of sample liquid were frozen and tests performed to time the length of irradiation until the sample was in a liquid state.

Test 1. Laser diode current 17A. The focal point of the beam was 3mm above the vial base, and the temperature of sample at commencement of test was minus 1WC. The elapsed time to liquid state was 105 seconds with a sample temperature of 450C Test 2. Laser diode current 15A. Other parameters were as in Test 1. The elapsed time to liquid state was 110 seconds with a sample temperature of 3 5'C.

These results show that a focused beam produces localised heating at the focal point within the sample. A preferred method would be a beam expand er/coll i mator to produce a 2mm diameter parallel beam to irradiate the sample cell. This would also remove the need for accurate height adjustment of the output optical assembly.

In a further test, a sample of horse blood was placed in a Microtitre sample tray (0.2mi). The rise of temperature within the sample was 10 times greater than the tests with water/food colour solution described above. Therefore, in order to determine the energy required to raise the temperature of a sample, defibrinated horse blood was used as the sample medium. The initial results obtained show that the energy absorption by blood at the 8 1 Onin wavelength is 1 7 very high. Readings taken with an energy meter show that the input energy requirement is in the order of 0.2SW.

One tray of 40mm deep 0.5m1 samples and one tray of 15mm deep 0.2mi samples were each prepared with a cell filled with horse blood and surrounding cells filled with water. Both trays were heat sealed with a foil membrane and were frozen to -700C.

Initial thawing tests were made on the 15mrn deep sample tray. The laser energy was set at 0.25W with the focal point 4mm into the sample. The time to thaw the sample to liquid was 8 minutes.

The 40mm sample tray was processed at the same energy level (0.25W). Thawing of the sample was not complete, the majority of the upper part of the sample remaining frozen.

The test was repeated with the laser energy increased to 2W. This caused an area at the base of the sample to coagulate and overheat and, after one minute, perforation damage was caused to the base of the sample cell.

With the energy reduced to 1.5W the result was less catastrophic, but nevertheless a small perforation occurred after 2 minutes. Further reduction of energy to LOW enabled thawing to be completed without damage to the cell, although examination of the sample revealed gas bubbles and coagulation of the blood at the base of the cell. At the end of the test the temperature at the cell base was 30'C.

8 Hence, the type of laser diode may be defined as having a maximum output of no more than lW, being a single emitter device coupled to the sample by a simple optic.

A further test was made with the laser energy at IW, modulating the laser energy at a 1: 1 mark/space ratio with the on time being 15 seconds. The sample start temperature was -50'C and examinations of the sample were made at 5 minute intervals. After 5 minutes thawing at the base of the sample had commenced to a depth of 3mm. At 10 minutes the thawed depth had increased to 8mm and there were no gas bubbles forming within the sample. At 15 minutes thaw depth was 12mm with no gas bubbles. At 20 minutes the sample surface had begun to thaw and after a further 1.5 minutes the sample was ready for extraction. Some ice crystals were evident on the walls of the cell (due to the surrounding cells remaining frozen).

Test completed show that the laser beam thawing of 0.2m1 sample cells is a viable proposition. Thawing of the 40mm deep, 0.5m1 sample size cells initially caused some problems but these were resolved by modulating the laser energy. In all test cases, adjacent cells remained frozen although a temperature rise due to the ambient air temperature was noticed.

Finally, tests were performed to detect any heating effect to cells of the sample tray adjacent to the cell undergoing heating and the temperature of adjacent cells was observed to rise by about PC for each WC of temperature increase in the sample. Further tests showed that temperature rise in adjacent cells was caused by two independent factors, namely:

1. The laser beam used for these tests has a focal point 25mm from the objective causing the beam spread to impinge on the adjacent cells.' and I- 9 2. The construction of the sample trays with webs between the cells allows conduction of heat. This effect becomes apparent between 2.5 and 3 minutes from the start of the test.

The first factor was most apparent on sample trays with a cell depth of 40mm, but use of an expander/collimator lens to obtain a parallel beam has been found to reduce this factor.

The results of these tests are shown graphically in Figure 1.

Claims

A method of retrieving an individual sample of biological material from a frozen assembly of a plurality of such samples, said method comprising the steps of applying energy from a laser source of energy to a single storage site within the assembly, monitoring the temperature of said site and, in dependence thereon, adjusting the power input to the laser source and/or the duration of input.
A method as claimed in claim 1, wherein the laser energy is pulsed.
A method as claimed in either claim 1 or claim 2, wherein the temperature of a particular sample is monitored by laser beam feedback.
A method as claimed in any one of the preceding claims comprising the further step, once the individual sample has been removed from its well, of replacing the assembly containing the remaining samples into cold storage without the remaining wells of the sample having been disturbed.
5.

A method as claimed in any one of the preceding claims, wherein the laser energy is directed towards a particular sample by relative movement along XY coordinates using stepper motors to move a laser generator, reflector means and/or the assembly.
6. A method of retrieving an individual sample of biological material from a frozen assembly of a plurality of such samples substantially as described herein with reference to the accompanying drawings.