Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/02—Form or structure of the vessel
  • C12M23/08—Flask, bottle or test tube
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
  • C12M41/14—Incubators; Climatic chambers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability

Definitions

• 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.
• neutrophil leukocytes 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the present invention resides in a microassay method for measuring cell adherence including the steps of:-
• the percentage of cells adhering to the fibrous material i.e. the percentage of "sticky” cells
• the percentage of cells collected at the other end of the tube i.e. the percentage of "sticky” cells
• the leukocytes are separated from the blood prior to step (a).
• the tube is placed in an incubator for a predetermined period, and at a pre- ' etermined temperature.
• 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.
• 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.
• 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.
• 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.
• the apparatus may include a timer means which is operable to apply the partial vacuum to the tube (or tubes) for a preset period.
• 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.
• test tube support plate 12 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
• 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.
• 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.
• 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.

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• 1983
  • 1983-10-11 WO PCT/AU1983/000145 patent/WO1984002532A1/en not_active Application Discontinuation
  • 1983-10-11 EP EP83903155A patent/EP0160640A1/de not_active Ceased

Patent Citations (2)

Non-Patent Citations (1)

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