JPH0117111B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is a method for separating leukocytes from human blood, and in detail,
mononuclear, using a separation medium consisting of a single aqueous solution of (i) a red blood cell aggregating agent or aggregating agent and (ii) a compound that is highly soluble in water and produces a solution of relatively high density, relatively low viscosity, and low permeability. A rapid one-step density separation method for red blood cells, polymorphonuclear leukocytes, and red blood cells. The inventors are aware of the many different techniques and methods used to separate and purify blood cell groups (and types within groups). One of the most common techniques used to separate white blood cells from red blood cells is to simply mix the blood sample with a solution that agglutinates the red blood cells, thereby increasing the sedimentation rate of the red blood cells. . The concentration of the separation liquid is such that it does not significantly affect the sedimentation of the leukocytes and allows the leukocytes to be collected from the top of the separation liquid when the red blood cells have sedimented. Newer developed techniques do not actually mix the hemagglutinating agent with the blood, but instead utilize a method in which the blood sample is carefully layered on top of a separation medium, and the red blood cells are agglutinated on the interface. Or aggregate and settle to the bottom of a test tube containing medium. Several types of polymer compounds are well known to agglutinate red blood cells, such as
FICOLL 400 (registered trademark of Pharmacia FAIN CHEMICALS GmbH, Sweden) is a high molecular weight polymer of neutral, highly branched white sugars, and these generally have a relatively high density and a relatively low viscosity. zoate or sodium
It is mixed with a compound in solution form, such as diatrizoate. Separation can be carried out at unit gravity or by centrifugation. The white blood cells mostly remain at the interface, but this conventional method has not been able to separate the types of white blood cells, namely mononuclear and polymorphonuclear leukocytes, at least in a one-step method using a single-density separation medium. . 1 to perform this separation
Two existing methods use Isopaque−Ficoll to isolate mononuclear cells.
® mixture (Isopaque solution obtained from Nyegaard, Norway, based on sodium metrizoate) in the first step, followed by dextran or gelatin to sediment the red blood cells. This method separates polymorphonuclear leukocytes. Other existing methods involve a first step of dextran precipitation to obtain mixed leukocytes, followed by a second step of centrifugation using Isopaque-Ficoll density gradient media to separate small groups or types of leukocytes. This method requires another operational step in which contaminated red blood cells are lysed with ammonium chloride in order to obtain relatively pure polymorphonuclear leukocytes. Alternatively, two or more types of separation solutions with different densities are stacked on top of each other,
The use of discontinuous density gradients is known. These densities are selected such that the desired range of (discontinuous) gradients is created. Another developed method that has been reported utilizes a one-step method, which was only effective in isolating single nuclei from whole blood samples. For example, it is known to use a separation medium consisting of a Hypaque-Ficoll mixture with a density of 1.077 g/ml on which a blood sample is layered (herein
Hypaque is a registered trademark).
In this case, when the blood was centrifuged, red blood cells and polymorphonuclear leukocytes settled to the bottom, while mononuclear cells formed a layer at the interface. A fraction of the separated mononuclear cells was collected separately by blotting the interface layer with a pipette. The principles of density separation of blood cell populations by centrifugation are already well known to those skilled in the art and therefore need not be discussed in detail here. By carefully adjusting both the ratio of separation medium and the concentration of hemagglutinating agent (weight (w)/total volume (v)), we
It has been discovered that white blood cell fractions (mononuclear cells) and PMN (polymorphonuclear leukocytes) and also red blood cell fractions can be separated. Therefore, it is a principal object of the present invention to present a rapid one-step method for the simultaneous separation of mononuclear cells, polymorphonuclear leukocytes and red blood cells from whole blood utilizing a single separating aqueous solution without discontinuous density gradients. It is a purpose. Therefore, according to the present invention, (i) mononuclear cells, (ii)
A one-step density separation method into fractions of polymorphonuclear leukocytes and (iii) red blood cells is presented, in which a whole blood sample is layered on an aqueous separation medium in a centrifuge tube;
The blood sample and separation medium are centrifuged to separate the whole blood sample into the aforementioned fractions, the separation medium comprising (a) an aqueous solution of a red blood cell aggregating or agglutinating agent, which is a high molecular weight saccharide or glucose polymer; and (b)
A mixture of aqueous solutions of water-soluble metrizoate or diatrizoate compounds with relatively high density, relatively low viscosity, and low permeability; (i) the density of this mixed solution of (a) and (b) is less than 1.095 g/ml at room temperature; and (ii) the concentration of (a) is in the range 5-11% (w/v % relative to the mixture). This method according to the invention has considerable advantages compared to conventional ones. First, this method is very simple and quick, taking only about 30 minutes to complete the entire operation;
Moreover, it can be carried out by an inexperienced person. Second
Relatively pure MN and PMN leukocytes are obtained. Third, both leukocytes are obtained in very high yields and the PMN fraction is not contaminated with red blood cells, thus avoiding ammonium chloride lysis and its expected deleterious effects on neutrophils. Finally, the immunological integrity of the cells is preserved, thus resulting in easier processing and faster separation, resulting in a healthier blood cell population (and type within the population). Very unexpected results have been obtained with the present invention. That is, the blood cells are separated into three separate fractions in one step, forming one band at the blood sample-separation medium interface, and an additional band just below this top band. The denser red blood cells form a precipitate. This uppermost zone contains mononuclear cells, while the lower zone contains relatively heavy cells.
Contains PMN white blood cells. In order to further illustrate and illustrate the invention, we provide below some examples which describe the manner in which the invention may be carried out. In these examples, the separation liquid medium is
Hypaque 85% solution (28.33% sodium 3,5-
Sterilization of diacetamide-2,4,6-triiodobenzoate and 56.67% n-methylglucamine 3,5-diacetamide-2,4,6-triiodobenzoate (purchased from Winthrop Laboratories, New South Wales, Australia) (aqueous solution) and Ficoll 400 (Palmacia, Sweden) dissolved in distilled water at a concentration of 10 w/v%. This separation medium is 20ml Hypaque 85%
and 90 ml of a 10% Ficoll aqueous solution, and the density of this mixture was 1.114 g/ml at room temperature. Sterile disposable plastic test tubes of 15 x 105 mm (diameter x length) were used for centrifugation. 3 ml of separation liquid was added to each tube. Next, 5-6 ml of heparinized whole human blood obtained by venipuncture was carefully layered on top of this separated solution using a pipette. At this time, the height of the blood column is 3.5cm.
It became. The test tubes were then spun down in a conventional centrifuge with a swing-out bag (in which it was supported) at 200 g for 20-30 minutes at room temperature. Of course, the centrifugation time and speed may be varied according to the common knowledge of those skilled in the art. After centrifugation, three distinct blood cell layers were observed. It has been demonstrated that the boundary layer consists of mononuclear cells, the middle layer polymorphonuclear leukocytes and the lower layer red blood cells that have settled to the bottom of the tube. The two leukocyte layers were then separately pipetted, washed three times, and resuspended in a known solvent. Table 1 shows the relative numbers of various white blood cells in the top two layers. These results are expressed as the mean ± standard deviation of the results of five experiments using (terminal) whole blood obtained from five different normal subjects.

[Table] The total leukocyte recovery rate was over 80% in all experiments, and the cell survival rate was also over 98%. It was found that the immunological functions of lymphocytes and neutrophils purified using this method remained intact. In another experiment conducted by the inventor, MN and
It was found that in order to satisfactorily separate PMN leukocytes, the density of the separation solution must be 1.095 g/ml or higher. As a result of repeated tests with separation solutions in which Ficoll was kept at a certain concentration and the density was changed from 1080 to 1.120g/ml, the density of the separation solution was 1.095g/ml.
It has been clearly established that sufficient separation of the cell fractions occurs only when . The distance between the MN and PMN fraction bands was found to be approximately proportional to the height of the blood sample (above the separation mixture) in the test tube. That is, the higher the height of the whole blood column, the greater the distance between each band. Tests also showed that when the blood sample in the test tube was kept at a certain height, the distance between the MN and PMN leukocyte zones remained approximately constant regardless of the diameter of the test tube. It has also been found that the concentration of the Ficoll aqueous solution must be 7% or higher (w/v % relative to the separation solution used), preferably 9%, but not higher than 11%. Technicians in this field will understand that Ficoll
It should be recognized that the concentration of can not be made too high. The density of Ficoll-Hypaque separation solution is 1.114g/
ml (room temperature) and Ficoll at 9% (w/v) gave the best results. In another experiment conducted by the inventor, it was used to suitably adjust the density of Ficoll solution.
It has been determined that the Hypaque 85% solution can be exchanged with other support media aqueous solutions such as sodium metrizoate, sodium diatrizoate, meglumine (N-methylglucamine) metrizoate or meglumine, diatrizoate and mixtures thereof. Meglumine diatrizoate or a mixed solution of meglumine metrizoate and sodium methozoate or diatrizoate (+Ficoll solution) can give a more clearly demarcated leukocyte fraction zone compared to Ficoll aqueous solution + unmixed sodium metrizoate or sodium ditrizoate solution. I understand. All of these support media are highly soluble in water, producing solutions of relatively high density and relatively low viscosity and low permeability. It is therefore suitable for mixing with Ficoll in order to prepare a dense solution without unduly affecting the final viscosity of the mixed separation aqueous solution (ie increasing the viscosity). Similar experiments conducted by the inventors have shown that the hemagglutinating agent in the separation medium can be an aqueous solution of dextran (dextran is a high molecular weight polymer of glucose with a molecular weight of up to about 370,000), which still binds blood cells to MNs and PMNs. It was found that the erythrocyte fraction was separated (however, the effect was relatively weak). However, it was found that the concentration range of dextran (molecular weight 264,000, Sigma Chemical Co., USA) that gave satisfactory results was slightly different from that of Ficoll 400, and it was found to be 7-11% of Ficoll in the Hypaque mixture. Actually 5-10 in comparison
It was between %. The density of the dextran-containing separation solution, as with the Ficoll mixture, must also be greater than 1.095 g/ml to produce the desired blood cell fraction. Summarizing each of the foregoing examples, it will be appreciated that the inventors have successfully created a rapid one-step process using a single separated mixed aqueous solution with density and concentration within certain specified limits. By this method, the diagnosis of leukocyte types for immunological purposes can be carried out very quickly, simply and effectively.

Claims (1)

[Claims] 1. A whole blood sample is layered on an aqueous separation medium in a centrifuge tube, and the blood sample and separation medium are centrifuged to separate the whole blood sample into mononuclear cells, polymorphonuclear leukocytes, and red blood cell fractions. the separation medium is a mixture of (a) an aqueous solution of a high molecular weight white sugar or glucose polymer red blood cell aggregating or agglutinating agent; (b) an aqueous solution of meglumine diatrizoate or meglumine metrizoate and an aqueous solution of sodium metrizoate or sodium diatrizoate; The density of the mixture of a) and (b) is 1.095 g/ml at room temperature.
In the above, (ii) whole blood characterized in that the concentration of (a) is in the range of 5 to 11% (w/v% with respect to the mixed liquid).
A rapid one-step blood density separation method for (i) mononuclear cells, (ii) polymorphonuclear leukocytes, and (iii) red blood cell fractions. 2. A blood density separation method according to claim 1, characterized in that (a) is a white sugar polymer, and the concentration of this white sugar polymer is in the range of 7 to 11% (w/v% relative to the solution). 3 In claim 2, the white sugar polymer has a molecular weight of about 400,000 in a rapid one-step process.
A blood density separation method characterized in that Ficoll (registered trademark). 4. The blood density separation method according to claim 1, wherein (a) is a glucose polymer, and the concentration thereof is in the range of 5 to 9% (w/v% with respect to the solution). 5. The blood density separation method according to claim 4, wherein the glucose polymer is dextran (registered trademark) having a molecular weight of about 264,000. 6 In claim 1, the separation medium has a concentration of 7 to 11% (w/v% relative to the solution).
An 85% aqueous solution of Ficoll and Hypaque, with a concentration of 1.095 to 1.20 g/ml at room temperature.
A blood density separation method characterized by being in the ml range. 7. The blood density separation method according to claim 1, wherein the solution density is 1.114 g/ml at room temperature.