Note: Descriptions are shown in the official language in which they were submitted.
1~;27~7
This invention relates -to a method of separation of the
leucocyte populations of human blood, and more particularly
to a rapid one-step density separation method of mono-
nuclear leucocytes, polymorphonuclear leucocytes and erythrocytes
5. (red blood cells) using a separating medium consisting of a
single aqueous solution of (i) an erythrocyte aggregating
or clumping agent and (ii) a compound which is highly soluble
in water and gives a solution of relatively high density and
relatively low viscosity and osmolarity.
10. BACKGROUND OF THE INVENTION
The Applicants are aware of a number of different
techniques and methods used in the separation and purification
of blood cell populations (and sub-populations). One of the
most commonly used techniques for separating the white blood
15. cells (leucocytes) from the red blood cells (the erythrocytes)
is to simply mix a sample of blood with a solution which
aggregates the red blood cells to thereby increase their
rate of sedimentation. The density of the separating fluid
is such that the sedimentation of the white blood cells is
- 20. only partially affected and can be collected from the upper
part of the separation fluid when the red blood cells have
- sedimented.
- A more recently developed technique makes use of a system
where the red blood cell aggregating agent is not actually
25. mixed with the blood but rather the blood sample is carefully
layered onto the top of the separating fluid medium where-
upon the red blood cells are caused to agglutinate or agg-
regate at the interface and sediment to the bottom of the
.
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tube in which the medium is placed. There are several well
known high polymer compounds which agglutinate the red blood
cells, for example FICOLL 400 (registered Trade Mark of
Pharmacia Fine Chemicals, Sweden) which is a neutral
5. highly branched, high molecular weight polymer of sucrose,
and these are generally mixed with a compound, in solution,
of relatively high density and relatively low viscosity, for
example sodium metrizoate or sodium diatrizoate. The
separation can be carried out at unit gravity or by
10. centrifugation. The majority of the white blood cells
remain at the interface but such previous systems have not
been effective in fractionating the white blood cell sub-
populations, namely the mononuclear and the polymorphonuclear
cells - at least not in a one-step process using a separating
15. medium of single density. In order to achieve such separation,
one known method involves the isolation of the mononuclear
white blood cells by centrifugation as a first step using a
Isopaque-Ficoll (registered Trade Mark) mixture (Isopaque
solution being obtained) from Nyegaard & Co. of Norway and
20. having as its main component sodium metrizoate) followed by
separation of the polymorphonuclear white blood cells by
using dextran or gelatin in order to sediment the red blood
cells. Other known methods have used dextran sedimentation
as a first step to obtain mixed white blood cells (leucocytes),
25. followed by a second step of centrifugation using a Isopaque-
Ficoll density gradient medium in order to separate the white
blood cell sub-populations. A further step is required to lyse
contaminating red blood cells with ammonium chloride in order
to obtain relatively pure polymorphonuclear cells. It is
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also known to make use of a discontinuous density gradient
where 2 or more separating solutions of different densities
are layered on top of one another. The densities are selected
so as to provide a (discontinuous) gradient over the desired
5, range.
In another reported development, a one-step process
was used but was only effective in separating the mononuclear
leucocytes from the whole blood sample. For example, it was
known to use a separation medium consisting of a mixture of
10. Hypaque-Ficoll having a density of 1.077 g/ml onto which the
blood sample was layered. Following centrifugation of the
blood, the red blood cells and the polymorphonuclear leucocytes
sedimented to the bottom while the mononuclear leucocytes
formed a band at the interface. The separated fraction of
15. mononuclear white blood cells was separately recovered by
pipetting off the interface layer. The principles of density
separation of the blood cell populations by the centrifugation
method is well known to those skilled in the art and hence
need not be discussed in any detail.
20. BRIEF SUMMARY OF THE APPLICANT'S INVENTION
.. ..
The Applicants have discovered that by carefully
controlling both the specific gravity of the separating
fluid medium, and the concentration of the erythrocyte
aggregating agent (based on weight/total volume) one is able
25. in a single step to isolate the M.N. (mononuclear) and P.M.N.
(polymorphonuclear) white blood cell fractions and also the
fraction of red blood cells.
It is the main object of this invention therefore to
provide a rapid one-step process for the simultaneous isolation
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of the mononuclear leucocytes, the polymorphonuclear leucocytes
- and erythrocytes from whole blood using a single separating
aqueous solution without a discontinuous density gradient.
According to this invention therefore, there is
5. provided a one-step method of density separation of whole
blood into (i) mononuclear leucocyte (ii) polymorphonuclear
leucocyte and (iii) erthrocyte fractions wherein a whole
blood sample is layered onto an aqueous separating medium
in a centrifuge tube and the blood sample and separating
10. medium centrifuged so that the whole blood sample is
fractionalised into the fractions hereinabove, wherein the
separating medium consists of a mixture of (a) an aqueous
solution of an erythrocyte aggregating or clumping agent being
a high molecular weight sucrose or glucose polymer and (b)
15. a solution of a water soluble metrizoate-or diatrizoate-
compound of relatively high density and relatively low
viscosity and osmolarity, wherein (i) the density of the
solution mixture (a) and (b) is greater than 1.095 gm/ml at
room temperature, and (ii) the concentration of (a) lies
20. in the range of 5 to 11% (based on the weight per total
volume of solution mixture).
The one step procedure according to this invention
has significant advantages over prior art methods. Firstly,
it is very simple and rapid requiring only some thirty
25. minutes for completion and may be performed by an inexperienced
person. Secondly, relatively pure populations of both MN
and PMN leucocytes are obtained. Thirdly, very high yields
of both white cell types are obtained and it has been shown
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that no red blood cell contamination of the PMN fraction
occurs so that ammonium chloride lysis and its possible
adverse effects on neutrophils is not required. Finally,
the immunological integrity of the cells is preserved and
5. hence one is able to obtain a much healthier cell (sub)
population as a result of less handling and more rapid
separation.
With this invention, a very unexpected result is
achieved - the blood cells are separated - by a one-step
10. process - into three distinct fractions, there being one
band at the blood sample - separating medium interface, a
further band just below the top band with the more dense
red blood cells forming the sediment. The top band contains
the mononuclear leucocytes whilst the band therebelow contains
15. the heavier PMN leucocytes cells.
In order to further describe and illustrate the present
; invention, we set out hereunder several examples which describe
the method by which the invention can be carried out.
In this example, a separating fluid medium is prepared
20. from a solution of Hypaque 85% (a sterile aqueous solution
of 28.33% sodium, 3,5-diacetamido-2,4,6-triiodoben-zoate,
and 56.67% n-methylglucamine 3,5-diacetamido 2,4,6 triiodoben-
zoate, purchased from Winthrop Laboratories, New South Wales,
Australia, and Ficoll 400 (Pharmacia, Sweden) dissolved
25. in distilled water at a concentration of 10% weight per
volume. The separating medium consisted of 20 mls of Hypaque
85% and 90 mls of 10% aqueous Ficoll, the density of the
mixture being 1.114 g/ml at room temperature.
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For the centrifugation process, sterile, disposable
15 x 105 mm (diameter:length) plastic tubes were used. To
each test tube was added 3 mls of the separating liquid medium.
Using a pipette, 5 to 6 mls of heparinised whole human
5. blood, obtained by vene-puncture, was then carefully layered
onto the top of the separating medium to give a blood column
height of 3.5 cm. The test tubes were then spun in a
; conventional centrifuge with swing-out buckets (in which the
tubes are supported) at 200 g for twenty to thirty minutes
10. at room temperature. Both the spinning time and speed can
of course be varied as is well known to one skilled in
the art. After centrifugation, 3 distinct blood cell layers
or bands were observed. The layer at the interface was shown
to consist of mononuclear leucocyte cells, the middle layer of
15. polymorphonuclear leucocyte cells, and the bottom layer of
erythrocytes which had sedimented to the bottom of the tube.
The two leucocyte bands were then removed separately with
different pipettes and washed thrice and resuspended in a
known medium. Table 1 shows the relative number of leucocyte
20. types in the upper two layers, the results being expressed
as the mean + standard deviation of 5 experiments using
whole (pheripheral) blood obtained from 5 different normal
individuals.
TABLE 1
25. CELL TYPES IN THE MONONUCLEAR AND POLYMORPHONUCLEAR CELL FRACTIONS
Cell Differential count (%)
Fractions
Lymphocytes Monocytes Basophils Neutrophils Eosinophils
Fr 1 83.9 + 1.6 13.8 + 2.3 0.5 + 0.5 1.8 + 0.8
(interface)
30. Fr 2 1.2 + 0.4 - - 96.4 + 1.0 2.4 + 1.0
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Total leucocyte recovery was greater than 80% of all
experiments and cell viability exceeded 98%. The immunological
function of the lymphocytes and neutrophils purified by this
procedure was shown to have remained intact.
5. Further experiments carried out by the applicants have
: shown that in order to get satisfactory separation of the MN
and PMN blood cells, the density of the separating solution
- must be greater than 1.095 gms per ml. Tests carried out
for separating solutions with densities ranging from 1.080
10. to 1.20 gm/ml at a given concentration of Ficoll 400 clearly
established that satisfactory separation of the cell fractions
occurred only in those instances where the density of the
separating solution exceeds 1.095 gms per ml.
The distance between the bands of MN and PMN fractions
15. was shown to be approximately proportional to the height of the
: blood sample (above the separation mixture) in the tube; that is, -
the greater the height of the whole blood column, the further
apart will be the bands. Tests also showed that, for a
given height of blood sample in a tube, the distance between
20. the bands of MN and PMN leucocytes remained approximately
constant for any tube diameter.
It was also shown that the concentration of the aqueous
Ficoll should be 7% or more (based on weight per total volume
of separating solution used) - preferably 9% but not more
25. than 11%. It will of course be appreciated by those skilled
in the art that the concentration of Ficoll cannot be too
high as highly viscous solutions effect the blood cell
functions and also cause clumping of the blood cells which
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has a deleterious effect on the fractionalisation process.
Best results have been obtained with a Ficoll-Hypaque
separating solution which has a density of 1.114 grams per
ml (at room temperature) and 9% (w/v) Ficoll.
5. Further experiments conducted by the Applicants established
that the Hypaque 85~ solution used to suitably adjust the
density of the Ficoll solution, can be replaced by other
supporting medium aqueous solutions such as sodium metrizoate
sodium diatrizoate, meglumine (N-methylglucamine) metrizoate
10. or meglumine diatrizoate, and mixtures thereof. Solution
mixtures of meglumine diatrizoate or meglumine metrizoate with
sodium metrizoate or diatrizoate (and the Ficoll solution)
were shown to give more clearly defined bands of leucocyte
fractions in comparison with a separating medium consisting
15. of aqueous Ficoll and an unmixed sodium metrizoate or sodium
diatrizoate solution. All of these supporting mediums are
highly soluble in water and give solutions of relatively
high density and relatively low viscosity and osmolarity and
- hence are suitable for admixture with Ficoll so as to produce
- 20. high density solutions without unduly affecting (i.e. in-
creasing) the final viscosity of the separating aqueous
mixture.
Similar experiments carried out by the Applicants have
shown that the erythrocyte aggregating agent can also be
25. aqueous Dextran (Dextran being a glucose polymer of high
molecular weight of up to about 370,000), in the separating
fluid medium and still fractionalise (but less effectively)
the blood cells into the MN and PMN leucocytes and erythrocyte
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fractions. However, the concentration range of the Dextran
(m.w. 264,000 - Sigma Chemical Co., U.S.A.), (based on
weight/total volume of separating solution), within which
satisfactory results were obtained, was found to differ
5. slightly to that of Ficoll 400, and in fact was between 5
and 10% as compared with 7 and 11~ for the Ficoll: Hypaque
mixture. The density of the Dextran-containing separating
solution, as with the Ficoll mixture, must also be above
1.095 gm/ml to produce the desired blood cell fractions.
10. A brief consideration of the above examples will
indicate that the Applicants have been successful in providing
a rapid one-step method using a single aqueous separating
mixture having density and concentration values within
certain prescribed limits. With this invention, one is
15. able to very quickly and easily and effectively diagnose
the sub-populations of the white blood cells for
immunological purposes.
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