WO1984002532A1

WO1984002532A1 - Microassay of cell adherence

Info

Publication number: WO1984002532A1
Application number: PCT/AU1983/000145
Authority: WO
Inventors: Yee Hing Thong; Joy Maree Currell
Original assignee: Univ Queensland
Priority date: 1982-12-22
Filing date: 1983-10-11
Publication date: 1984-07-05
Also published as: EP0160640A1; EP0160640A4

Abstract

A method for the microassay measurement of the cell adherence of leukocytes, lymphocytes, monocytes, neutrophils and like cells. The cells are mixed in a known concentration in a medium and a small sample (of 0.1 ml) is placed in contact with a predetermined weight or volume of inert fibrous material (17) in a small tapered tube or micro-column (18). During a predetermined contact time between the cells and the fibrous material (17), the tube is heated in an incubator for a predetermined period at a predetermined temperature. The tube or micro-column (18) is received in a test tube cap (15) to which is fitted a test tube (16). A vacuum pump (24) is connected to the interior of the test tube (16) and effluent (26) is collected in the test tube (16). The cell adherence is calculated by subtracting the percentage of cells in the effluent (26) from 100%.

Description

Title: "MICROASSAY OF CELL ADHERENCE"

BACKGROUND OF THE INVENTION (1) Field of the Invention

This invention relates to a method of, and apparatus for, the microassay of cell adherence. The invention is particularly suitable for, but not limited to, measuring neutrophil adherence. (2) Description of the Prior Art

The adherence of neutrophil leukocytes to vascular endothelium constitutes an important early step in a complex sequence of events which leads to a congreg¬ ation of these cells at sites of infection or inflammation.

It has been observed that the leukocytes become "sticky" (i.e. their adherence increases) when they receive a signal that bacteria is attacking the body cells. The leukocytes attach themselves to the walls of the blood vessels and migrate through the tissue to the infection sites to attack the bacteria.

If the adherence level of the leukocytes does not increase on receipt of the signal, they continue past the infection site, carried along in the bloodstream. With¬ out this defence directed against them, the bacteria rapidly multiply unchecked and the infection can spread rapidly through the body, leading to septicaemia. In a healthy person, approximately 60-90% of the leukocytes will display this increased adherence level when infection invades the body cells. Levels below that range indicate a problem has arisen and remedial action is necessary, while a level below 20% indicates that the problem is very severe and the person's body has little, if any defence, against any infection.

Once this phenomena became recognised, the problem arose how to accurately measure the adherence level, particularly in small children or the ill where only small blood samples may be available. One measuring technique has been to take a blood sample, separate the leukocytes (or white cells) , and place them in a dish. After a predetermined period, the leukocytes are washed from the dish and a count is made of the remaining leukocytes to determine the percentage of leukocytes with the necessary adherence level.

A second measuring technique has been to separate the leukocytes from the blood sample, place a predetermined weight of nylon fibre in a large test tube, pour the leuko- cytes into the test tube, allow the percolation of the leukocytes through the fibres for a predetermined (variable) time and measure the volume of leukocytes collected in the bottom of the test tube, subtracting this volume from the initial volume to calculate the percentage of leukocytes with the desired adherence level.

Both methods are unacceptable for measuring the leukocyte adherence of children as a very large blood sample e.g. 100ml. is required. In addition the second method is slow and surface tension may cause extra leukocytes to be held in the nylon fibre, resulting in a lower volume collecte in the bottom of the test tube and a too high adherence level reading.

SUMMARY OF THE PRESENT INVENTION It is an object of the present invention to provide a technique where very few cells are required to achieve an accurate result.

It is a preferred object to provide a technique where the number of cells required may be reduced by a factor of 10². It is a further preferred object to provide a method where surface tension effects and/or capillary effects are overcome.

It is a further preferred object to provide a method which is much faster than known techniques. it is a still further preferred object to provide a simple, yet effective apparatus suitable for carrying out the method.

Other preferred objects of the present invention will become apparent from the following descripti In one aspect the present invention resides in a microassay method for measuring cell adherence including the steps of:-

(a) placing a predetermined weight or volume of fibrous material in a small tube; (b) placing a predetermined weight or volume cell sample in the one end of the tube;

(c) applying at least a partial vacuum to the other end of the tube after a predetermined contact time between the cells in the cell sample and the fibrous material; (d) collecting the cells received at the other end of the tube which have passed through the fibrous material; and

(e) calculating the percentage of cells adhering to the fibrous material from the percentage of cells collected at the other end of the tube.

The percentage of cells adhering to the fibrous material (i.e. the percentage of "sticky" cells) equals 100% minus the percentage of cells collected at the other end of the tube. Preferably where the cells are leukocytes, the leukocytes are separated from the blood prior to step (a).

Preferably after step (b) , the tube is placed in an incubator for a predetermined period, and at a pre- ^' etermined temperature. In a second aspect the present invention resides

•in an apparatus for a microassay method of measuring cell adherence including:- a small tube containing a predetermined weight or volume of fibrous material; means to enable a cell sample to be placed at one end of the.tube; means connecting the other end of the tube to a source of at least a partial vacuum; means at the other end of the tube to collect the percentage of cells which pass through the fibrous material in the tube when the partial vacuum is applied to the tube.

Preferably the small tube is tapered towards its other end to retain the fibrous material in the tube. A suitable type of tube is the tip of a disposable pipette.

The fibrous material may include nylon fibres or any other fibres which are inert in the cells being measured.

The source of partial vacuum may include a simple vacuum pump. Preferably the tube is connected to the pump via a vacuum tank or manifold to damp any pulses in the vacuum pressure level in the tube.

Preferably the tube is sealed to the mouth of a disposable test tube by a test tube cap and the interior of the test tube is connected to the vacuum pump, tank or manifold to apply the vacuum to the lower end of the tube, the cells being applied to the upper end of the tube.

Preferably three or more cell samples are tested against a control sample, and the average of the measurements of the three samples is used to determine the adherence level of the cells . The apparatus may include a timer means which is operable to apply the partial vacuum to the tube (or tubes) for a preset period.

BRIEF DESCRIPTION OF THE DRAWING To enable the invention to be fully understood, a preferred embodiment will now be described with reference to the accompanying drawing which shows a simple laboratory apparatus, parts being shown in section for clarity.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT The apparatus has a test tube holder 10 formed of perspex. The holder has a base 11, test tube support plate 12 and tube guide plate 13 interconnected by end walls 14.

A plurality of holes are formed in the test tube support plate 12 and a test tube cap 15 is fixed in each hole. Each cap is adapted to receive and releasably support a small disposable test tube 16.

A pair of holes are formed in each test tube cap 15. A predetermined weight e.g. lOmg. of teased nylon fibres 17 is placed in a disposable micro-column 18

(comprising a pipette tip) and the micro-column 18 is plac in one of the holes of each cap 15. The nylon fibre micro column 18 may accommodate 0.1 ml. of the leukocyte contain ing medium which is sufficient for the microassay techniqu to be effected.

A plastic tubing 19 is fitted to the second hole in each test tube cap 15 and the other end of the tubing passes through a respective hole formed in a rubber plug 20 fitted to a tapered glass flask 21 which has an exhaust tube 22 extending to one side. This exhaust tube 22 is connected to the suction inlet 23 of a small vacuum pump 24 via a length of plastic tubing 25. The flask 21 acts as a vacuum manifold and damps any pulses in the vacuum level in the apparatus. The operation of the apparatus will now be described.

Purified leukocytes in medium 199 containing 5% autologous plasma were adjusted to a concentration of between 4 to 6 x 10 6ml —1. This concentration has been found to be suitable for use with nylon fibre columns weighing lOmg. The 5% autologous plasma enables optimal adherence of the leukocytes to occur.

0.1ml. of the leukocyte-containing solution is placed in each pre-heated micro-column 19 and the micro- - column is placed in an incubator at 37°C with a contact ti

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" of 5 minutes between the cells and the fibre. The micro- columns 18 are fitted^'.to the test tube caps 15 and the vacuum pump 24 is operated for 1-2 minutes, the vacuum pressure in the test tubes 16 being approximately l-2mm 5 Hg., and the effluent 26 is collected in the test tubes

16. The effluent 26 is removed and the leukocyte concen¬ tration in the effluent 26 is determined e.g. by counting in a Neubauer Haemocytometer or a Coulter Counter. The result is calculated as follows:- i _<= -I ,--. t nn (Leukocyte cone, in effluent „ -inr

¹⁰ % Adherence = 100 - _l (L_τ _e„u-kmocy,3t,_e c -o-n„e-—, i τ^~n^{~ x 10}°).

( original solution )

Preferably three samples from a particular leukocyte sample are taken and compared against a known control sample, the average of the three samples being used

15 to determine the % adherence of the leukocyte sample.

The control time between the cells and the fibre, the incubation temperature, the vacuum pressure in the test tubes and the length of application of the vacuum can be varied to suit the particular cells being tested. For

20 example, with leukocytes, best results have been obtained when the incubation temperature has been approximately 37 (which corresponds to normal human body temperature) .

As the vacuum in the test tubes 16 overcomes the surface tension and/or capillary effects in the micro-column

25 18, the results obtained are much more accurate than with known methods and yet a sample one-hundred times smaller than previously used is only required, making the technique suitable for the measuring of cell adherence of a child's blood sample.

30 While the embodiment described above refers to the measurement of cell adherence for leukocytes, it can be readily applied to the measurement of the adherence of other cells. For example, lymphocytes which have come into contact with cancer, will become "less sticky" when

35 brought into contact with a cancer antigen if they have previously encountered that antigen. This results in a higher count of lymphocytes in the effluent in the test tubes as less lymphocytes adhere to the nylon fibre. This increased level of lymphocytes in the effluent (or reduced adherence) is an indication of the presence of cancer in the body of the donor. The method and apparatu may also be used to measure the cell adherence of monocyte and neutrophils in a similar manner.

The apparatus described and illustrated is only a simple laboratory apparatus but the invention can be incorporated in semi-or-fully automated machines which introduce the cell-containing solution to the columns, incubate the columns and apply the vacuum to the columns. Such machines may also include the cell counters to give a read-out of the cell adherence.

Various changes and modifications may be made to the embodiment described without departing from the sco of the present invention as defined in the appended claims

Claims

1_. A microassay method for measuring cell adherence including the steps of:-

(a) placing a predetermined weight or volume of fibrous material in a small tube;

(b) placing a predetermined weight or volume cell sample in the one end of the tube;

(c) applying at least a partial vacuum to the other end of the tube after a predetermined contact time between the cells in the cell sample and the fibrous material;

(d) collecting the cells received at the other end of the tube which have passed through the fibrous material; and

2. A method as claimed in Claim 1 wherein:- the cells are separated from the blood and mixed in a medium prior to step (a) .

3. A method as claimed in Claim 2 wherein:- the medium contains a plasma and the concen¬ tration of cells is adjusted to fall within a predeter¬ mined concentration range.

4. A method as claimed in any one of Claims 1 to

3 wherein:- the tube is placed in an incubator for a predetermined period, and at a predetermined temperature, during the contact time between the cells and the fibrous material.

5. A method as claimed in any one of Claims 1 to

4 wherein:- the partial vacuum is applied to the tube for a predetermined period in a predetermined vacuum pressure level range.

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6. A method as claimed in any one of Claims 1 to 5 wherein:- at least three cell samples are tested against a control sample, and the average of the measurements of the test samples is used to determine the adherence level of the cel

7. An apparatus for the microassay method of Claims 1 to 6 including:- a small tube containing a predetermined weight or volume of fibrous material; means to enable a cell sample to be placed at one end of the tube; means connecting the other end of the tube to a source of at least a partial vacuum; means at the other end of the tube to collect the percentage of cells which pass through the fibrous material in the tube when the partial vacuum is applied to the tube.

8. Apparatus as claimed in Claim 7 wherein:- the small tube is tapered towards its lower end to retain the fibrous material in the tube; the fibrous material including nylon or other fibres inert in the cells being tested.

9. Apparatus as claimed in Claim 7 or Claim 8 wherein:- the source of vacuum is a vacuum pump; the tube being connected to the vacuum pump via a vacuum tank or manifold to damp any pulses in the vacuum pressure level in the tube.

10. Apparatus as claimed in any one of claims 7 to 9 wherein:- the tube is sealed to the mouth of a disposable test tube by a test tube cap and the interior of the test tube is connected to the vacuum pump, tank or manifold to apply the vacuum to the lower end of the tube, the cells being applied to the upper end of the tube.

11. Apparatus as claimed in any one of Claims 7 to

10 and further including:- an incubator to heat the cells in the tube to a predetermined temperature.