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Description  |
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This invention relates to methods of preparing and analysing particulate
matter of biological origin by luminescence detection.
There are many fluids both biological and non-biological which contain
substances which may require analysis either using a luminometer or other
luminescence detection methods. The fluids may be biological e.g. blood,
blood components, milk, colostrum, urine, tissue and tumour disaggregates
and exudates, lymph, ascites fluid, cerebrospinal fluid, bile and other
secretions and excretions of living organisms or non-biological e.g. fresh
water (rivers, lakes, ponds), sea water, industrial effluent, leachates.
The substances may be particulates of cellular or non-cellular varieties
such as leucocytes, platelets, components from tissue disaggregates,
components from tumours, protozoa, small invertebrates, algae including
blue greens, gametes and generally any other particulate material of a
biological origin which can be separated from its carrier fluid.
Much information can be obtained for example from measuring light emission
from particles such as leucocytes. This light emission can be enhanced by
adding luminogenic materials such as Pholasin (TM) and light can be
measured in various ways. Pholasin reacts with oxygen radicals and other
oxidants to produce light and leucocytes and other cells produce oxygen
radicals when activated. There is a rapidly growing interest in the
involvement of oxygen radicals in oncology, cardiology, rheumatology,
asthma, ageing, autoimmune diseases and diabetes mellitus for example. In
addition, there are the potentially damaging effects of cells activated
during renal dialysis or reperfusion of organs during surgery. Other areas
include study and assessment of disease activity in inflammatory diseases
and for distinguishing between inflammation and infection, these areas
being of particular interest to rheumatologists and haemotologists. A
quantitative measure of particulate activity, for example the activity of
leucocytes, would therefore be a valuable diagnostic tool.
In order to obtain quantitative results that can be used in diagnostic and
comparative studies, it is essential that: a constant proportion of
certain particles (preferably 100%) is removed from a fixed volume of
fluid which might contain a mixture of particles; that the particles are
not damaged in the process; that whatever treatment the particles
experience during any manipulation can be repeated in an identical fashion
on particles from another sample of fluid; that all parameters can be
controlled so that the results can be compared and related to known
factors.
There are known methods for separating particles, such as leucocytes, from
blood, for example, for subsequent use in analysis. Such methods include
multistep procedures including sedimentation with dextran, followed by
separation on density gradients. These procedures involve centrifugation,
mixing, incubating and sometimes lysis of unwanted red blood cells. They
take a number of hours to complete, involve skilled operatives and subject
the cells to uncontrollable variables which may inadvertently affect their
subsequent response to analytical procedures (Boyum, A. (1968) Isolation
of mononuclear cells and granulocytes from human blood. Scand. J. Clin.
Lab. Invest. 21, Supple 97 (paper IV), 77-89). In addition the process has
to be conducted in a laboratory and thus cannot be used in general medical
or veterinary practice, at the bedside, in the field or in an outpatient
clinic for example.
In response to the need for a rapid, simple and reliable method of
isolating leucocytes from whole blood a simpler method was introduced
(Ferrante, A. and Thong, Y. H. (1980) Optimal conditions for simultaneous
purification of mononuclear and polymorphonuclear leucocytes from human
peripheral blood by the HypaqueFicoll method. J Immunol Methods. 35,
109117), which involved layering whole blood on to a mixture of Ficoll and
sodium and/or meglumine diatrizoate prepared to specific densities,
centrifuging the tube and collecting layers of leucocytes which were
washed with centrifugation 2 to 3 more times. This `improved` method
which, enabled leucocytes to be separated, washed and ready for analysis
in about 1 to 2 hours still required a skilled operative and the need for
a centrifuge. Also, it was not possible to carry out the procedure
simultaneously on more than a very few samples (less than 4) and the
method is not suitable for most bloods other than human, does not work
efficiently on blood from people with, for example: juvenile rheumatoid
arthritis, microcytic hypochromic anaemia. The method may fail or give
variable results if an individual was receiving aspirin, indomethacin,
prednisone or aurothioglucose, or other drugs in the treatment of
bronchial congestion, immunodeficiency anaemia and other diseases. An
improved method, designed to enable leucocytes to be separated from these
`difficult` bloods was developed (Ferrante, A., James, D. W., Betts, W. H.
and Cleland, L. G. (1982) Rapid singlestep method for purification of
polymorphonuclear leucocytes from blood of patients with rheumatoid
arthritis. Clin exp. Immunol 47 749752) in which the viscosity of the
density medium was changed. The results of this improved method are still
variable and not useful for quantifiable and comparative results. In all
the methods, even the improved ones, the cells are subjected at times to
adverse conditions. And while it might be theoretically possible for a
trained operative to work in precisely the same manner at each separation,
the differences in the blood make it impossible for the blood from
different people and at different states of a disease to behave in
precisely the same manner. And it is impracticable, even for trained
operatives, to standardise the ways they perform the various manipulations
involved in the procedure. The method is therefore only suited to the
separation of leucocytes from whole normal blood.
Pall Corporation in U.S. Pat. No. 4,925,572 and U.S. Pat. No. 4,880,548,
disclose filters for depleting the leucocyte concentration of whole blood
and for reducing the concentration of leucocytes from platelet
concentrates. These filters are used on-line during blood or platelet
transfusions, whereby the filtrate which contains the red blood cells or
the platelets is allowed to enter the circulation of the patient while the
leucocytes are retained by the membrane.
According to a first aspect of the present invention there is provided a
method of analysing particulate matter of biological origin comprising the
steps of passing a fluid containing said particulate matter through a
membrane which membrane is adapted to hold said particulate matter and
subjecting said membrane together with said held particulate matter to
analysis by luminescence detection.
Preferably said analysis involves a luminometer and said membrane may be
placed in a luminometer cuvette or a microtitre plate prior to insertion
into the luminometer.
In the method leucocytes and/or platelets from a known volume of whole
blood are separated simply and rapidly from the rest of the blood
components on to a filter support from which useful, quantitative and
comparative measurements of light emission can be made. The measurement of
light, or other parameters, can be made a very short time, preferably
within two minutes, after collection of blood. The procedure can be
performed on blood from people excluded from the methods described above
and also from blood from nonhuman species, which also do not separate
properly using the above improved methods. The fluid from which the
leucocytes are separated can in addition to blood be milk, urine,
cerebrospinal fluid and any other fluid in which such particles are found.
The method can also be applied to the separation of particles other than
leucocytes and any substances that adheres to the membrane and can be
analysed.
In a preferred method there is included the step of treating the
particulate matter held by the membrane with a luminescent material which
reacts with certain chemicals in or produced by the particulate matter. It
is also possible that more than one luminescent material is used and these
luminescent materials may be applied before or after the membrane is
inserted in the luminometer.
In preferred methods said certain chemicals are radicals, such as
superoxide, O.sub.2 and the luminescent materials may be chosen from the
from the following examples, PHOLASIN, luminol, lucigenin. Other chemicals
such as ATP react with firefly luciferin plus firefly luciferase whereas
bacterial luciferase is the preferred luminogenic reagent when analysing
for NADPH and NADH.
Conveniently, the particulate matter held by the membrane is washed prior
to being treated with said one or more luminescent materials. Examples of
the washing substances are buffered or unbuffered salt solutions, blood
serum, plasma. In a further embodiment the fluid passes through other
membranes, each of which is adapted to prevent the through flow of other
preselected substances.
Preferably the fluid passed through the filter by gravity or increased
pressure on the fluid or reduced pressure on the downstream side or a
combination of these, or by the capillary attraction of an absorptive pad
held against the underside of the filter.
According to a second aspect of the present invention there is provided a
filter device for filtering a fluid containing particulate matter of
biological origin, said device containing a membrane through which, in
use, the fluid is passed to leave the particulate matter, the membrane
being adapted for use in luminescence detection apparatus to enable the
particulate matter to be analysed.
Preferably the membrane is removable from the device for use with the
luminescence detection apparatus.
In a preferred embodiment the membrane is adapted to remove said
particulate matter by adsorption and has a Critical Wetting Surface
Tension greater than 53 dynes/cm.
Another arrangement has the membrane held between two separable parts.
Conveniently the membrane sits on a perforated support and may have its
edges sealed or clamped between upper and lower frames.
Clearly the device may have more than one membrane for removing different
substances from the fluid.
According to a third aspect of the present invention there is provided a
syringe comprising a barrel having a fluid inlet, a plunger slidably
disposed in the barrel and means for piercing the plunger whereby, in use,
after filling the plunger can be pierced to allow the liquid in the
syringe to be exposed to the atmosphere at the plunger as well as at the
fluid inlet and thus to allow liquid to flow from the syringe without
moving the plunger axially relative to the barrel.
Preferably the plunger has a rod connected to it by which the plunger is
moved and the piercing means comprises a pin slidably mounted coaxially
within said rod. Also the rod may have a screw thread at its distal end
engaging in a matching screw thread in the plunger. Rotation of the rod
allows the controlled exposure of the contents of the syringe to the
atmosphere at the plunger end.
Embodiments of the invention will now be described in more detail. The
description makes reference to the accompanying drawings in which:
FIG. 1(a) is a sectional view of a filter arrangement according to an
aspect of the present invention.
FIG. 1(b) is a sectional view of an alternative part of the filter
arrangement shown in FIG. 1(a).
FIGS. 2(a) to (c) show various types of membrane for use in the filter
arrangements,
FIGS. 3(a) to (c) are diagrammatic section views of a syringe according to
other aspects of the present invention,
FIG. 4 is a perspective view from above of a further filter arrangement
according to the present invention, and
FIG. 5 is a perspective view from below of part of a still further filter
arrangement according to the present invention.
It is often necessary to remove substances, such as cellular and/or
non-cellular particulates or other dissolved substances, from a carrier
fluid which may be biological or non-biological, so that the substance can
be analysed. The fluids are collected in a number of ways which could
range from a syringe to a beaker.
The fluid is then passed through a filter arrangement 10 as shown. In FIG.
1(a) the filter arrangement 10 comprises a funnel 11 having a perforated
support 12 on which sits a membrane 13 the latter having a greater
diameter than that of the perforated plate. A tubular reservoir 14 is
attached to the funnel for gravity filtration of the fluid and the size of
the membrane 13 is such that it is held in position by the reservoir 14
when the reservoir is screwed into place. In FIG. 1(b) there is shown an
alternative reservoir provided with a Luer fitting 15. As will be readily
appreciated the fluid is able to pass through the membrane either under
gravity or by applying an increased pressure to the liquid or by reducing
the pressure on the downstream side of the membrane. Clearly it is also
possible to use a combination of these techniques as is well known.
More than one membrane could be employed, the purpose of such a technique
being to collect different substances on different membranes. The
construction of such a modification is not described because it clearly
involves putting the membranes downstream from one another. Similarly each
membrane can be made up of a number of layers of suitable materials.
The membranes themselves will not be discussed in detail. Membranes or
other filters are well known to remove certain substances so as to purify
the fluid which passes through the filter unhindered. however, Pall
Corporation have invented certain membranes for using in obtaining blood
and platelet concentrate which is free of leucocytes which are retained on
the filter membrane by adsorption. However, bacteria and other substances
are retained by careful regulation of the pore sizes in the membrane, less
than 0.2 um in the case of bacteria.
With the FIG. 1 arrangements in which the membrane 13 is removable, the
membrane will ideally be supplied in the form of individual units of the
correct shape. This may be circular, rectangular or any other suitable
shape. If a number of layers of filter material are used for each membrane
then they may be left loose at the edges (FIG. 2(a)) or they may be sealed
in some suitable way for example by melting, by using a filler 18 FIG.
2(b) or by clamping the edges between two frames 19 (FIG. 2(c)).
Once the fluid has been filtered, the substance may be washed so as to
remove any part of the fluid which could interfere with subsequent use of
the membrane. Such washing fluids may include buffered or unbuffered salt
solutions or biological fluids such as blood serum or plasma.
One particular type of analysis which is of interest is luminometry
although it will be realised that many other methods of analysis are
envisaged.
Where the membrane is removable from the filter arrangement the membrane is
then placed in a luminometer cuvette or microtitre plate or any other
suitable receptable from which light can be detected. When inserted into
the luminometer the upstream side of the membrane should face the light
detector in the luminometer. If using a microtitre plate, the membrane
should conform to the shape of the wells in the plate.
When measuring light emitted from the substance retained by the membrane,
the membrane is treated or immersed in a base liquid which includes one or
more luminescent materials such as PHOLASIN (Trade Mark), luminol,
lucigenin, firely luciferase plus firely luciferin, and bacterial
luciferase for example. The principles of analysis by luminescence are
well known. Luminescent materials emit light in response to certain
chemicals. One such chemical which stimulates PHOLASIN is superoxide,
O.sub.2.sup.- radical. Obviously the luminescent material chosen depends
on the features which you wish to detect and analyse. More than one
luminescent material can be present in the base liquid if desired so as to
monitor different chemicals.
Various measurements can then be taken on the luminometer such as the
resting glow of the luminescent material, the glow caused by metabolites
of the substance under analysis, the resting glow of other luminescent
materials together with any glow caused by metabolites.
Also, other substances could be introduced to the membrane whilst in the
luminometer so as to activate certain other chemicals which could result
in a different glow for measurement. For example, leucocytes are activated
by a number of substances such as phorbol myristate acetate, tumor
necrosis factor, opsonised serum, opsonised zymosan, a suspension of latex
particles. The luminometer can be used to monitor the progress of the
reactions.
The measurements made can give diagnostic indications of the physiological
or pathological state of a patient. The membranes and the substances
retained thereon are ready for analysis within minutes of the collection
of the fluid. The process can readily be conducted in situ and therefore
removes the expensive and time consuming step of sending the fluid to a
specialised laboratory.
The apparatus could be supplied in kit form comprising say a syringe to
obtain the fluid, a filter arrangement to isolate the substance of
interest on the membrane and luminescent material to enable the membrane
to be treated after insertion into a luminometer, portable luminometers
being readily available. Clearly however suitable adaptions will be
necessary to enable the kit to be used with all commercially available
luminometers.
A further filter arrangement is shown in FIG. 4 and comprises a piece of
membrane 13 which is attached (removably or immovably) to a support. The
support may be in the form of a rigid strip 20 with rectangular holes cut
in it at intervals and below which is attached the membrane. Other hole
shapes are of course possible.
The support 20 may have an extension in the form of a flange 22 adapted to
be held by forceps. The flange can be removed by cutting when the strip
has been placed in the luminometer cuvette, thus obviating the need for
the strip to be handled in any way.
At the end of the filtration, and subsequent washing of the membrane 13, i)
if the membrane were removably attached to its support, the membrane would
be removed from its support, perhaps by peeling, and placed in the
luminometer cuvette; ii) if the membrane were immovably attached to its
support, the support would be cut along guide lines 23 in such a way as to
free a section of it containing the membrane from the rest of the support
and this section would be placed in the luminometer cuvette.
A relatively thick absorptive pad 24 is held under the filter assembly so
that fluid added to the membrane would pass through the membrane, after
leaving the desired components in or on the membrane, and be absorbed by
capillary attraction into the absorptive pad 24.
The absorptive pad 24 would itself be contained in a structure or enclosure
25 that would allow none of the absorbed fluid to spill from the sides or
bottom of the pad other than into this enclosure 25. Alternatively this
structure could form an integral part of the absorptive pad, such that the
pad 24 is formed with impermeable side and bottom walls.
In another arrangement (not shown) the membrane, or sections of the
membrane, may be held between two layers of supporting material in which
are formed opposed holes for the passage of the fluid through the membrane
and into the absorptive pad. In this case the appropriate piece of
membrane would be removed from between the sandwich after filtration and
washing and placed in the luminometer cuvette.
In another possible arrangement shown in FIG. 5 the support 20 may be in
the form of a strip 20 containing a number of wells 26, perhaps six. FIG.
5 is of such a structure and shows one well viewed from underneath, this
figure is to no particular scale. Each well has a hole 21, perhaps
rectangular, at the bottom, below which the membrane 13 is attached.
If the luminometry is to be carried out in a micotitre plate luminometer
the filtration device may consist of a microtitre plate strip or block in
which the base of each well is furnished with a piece of the membrane as
in the other devices described above. After filtration and washing of the
samples, the strip or block is placed into a similar but shorter strip or
block with the bottoms of the wells entire so that further flow of fluid
through the membrane is prevented. This compound structure is then placed
in the microtitre plate reader, perhaps after the addition of appropriate
reagents to each of the wells.
The techniques described can also be used to test milk for various reasons.
One reason is to analyse the leucocytes which are present in milk to gain
information about certain diseases in the animals, for example mastitis in
cows. Milk producers can claim a premium if their product reaches certain
standards. Data can therefore be obtained almost instantaneously in situ,
without the need for skilled labor. Such techniques will also have obvious
benefits in third world countries where alternative facilities for testing
may not be available.
Also milk can be tested in this way to assess under--or
over--pasteurisation by for example analysing the concentration of various
enzymes. Bacterial contamination could also be monitored at the same time.
Another application for this invention is for the rapid determination of
leucocytes in urine as part of a luminescent urinary tract infection
screening kit. The activity of any leucocytes present in the urine and
adsorbed on to the membrane will be assessed with the luminogenic reagent
Pholasin or other suitable reagents. The invention will provide a simple,
rapid measure of pyuria and its relation to bacteriuria. It will lead to
simplification of the luminescent test for determining bacteria in urine,
eliminating the requirement to destroy any ATP from somatic cells which
might be present. Elimination of such a step will increase both the speed
and reliability of that test.
FIG. 3(a) shows a syringe 30 having a barrel 31 and an inlet 32 through
which liquid is drawn when a plunger 33 is withdrawn. The plunger 33 being
pulled back by rod 34 attached thereto. A pin 35 is disposed within the
rod 34 so that when the syringe 30 has been filled, the plunger can be
pierced. The fluid in the syringe can then flow out under gravity. This is
advantageous because it is not always desirable to eject the fluid under
pressure by depressing the plunger. Clearly other ways of exposing the
fluid at the plunger end to atmospheric pressure, for example the rod 34
may be attached to the plunger 33 by means of a screw thread passing
through the plunger as shown in FIG. 3(b) by means of a bayonet fitting
plunger are possible and need not be confined within the rod 34.
FIG. 3(c) shows another syringe 30 in which the rod 34 is in the form of a
cylinder which surrounds the pin 35. In use, when the syringe is held
vertically so that the contents can be allowed to flow out at a rate
controlled by the pin 35, another fluid is introduced into the cylinder
34. If the second fluid is of lower density than the contents of the
syringe it will flow into the barrel and flush out the barrel of the
syringe. When this second liquid impinges on the filter it will act as a
washing liquid to wash away any substances not intended to be retained by
the filter material. A similar adaptation could be made to the other
syringe embodiments mentioned.
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