FR2461951A1 - Preparation of slide mounted blood sample - including exposure to fixing agent to maintain cell morphology - Google Patents

Abstract

A blood specimen consisting of a monolayer of sepd. blood cells is prepd. on a slide suitable for automated analysis. A sample is spun on the slide about an axis perpendicular to a support for a short time to develop a monolayer. This separates the red blood cells and throws excess cells radially outwards to form an evenly distributed monolayer. This monolayer is subjected to a fixing agent subsequent to the spinning operation and prior to drying. This fixes the natural shapes of the cells before they flatten during drying. The slide is finally dried. The method is used to prepare a blood cell specimen for diagnostic analysis. The cells are fixed during a critical step of their preparation so that their morphology is preserved for later analysis.

Description

The present invention relates to a method and apparatus for preparing blood cell specimens for diagnostic analysis. More specifically, it aims to fix or preserve the morphology of the cells with a view to subsequent automatic analysis.

 The invention relates in particular to the fixation carried out on the cells, during a precise and critical stage of the preparation of the samples, in order to preserve their morphology with a view to subsequent analysis. This minimizes the distortions that appear with other methods of preparing samples with cells carried by a micro-skeptic blade. Without the invention being limited to a particular kind of cells or samples, we will refer to describe it for the rapid preparation of red blood cells for automatic analysis. One of the changes in aspects noted so far now, with the known modes of preparation of microscopic slides, is a morphological alteration suffered by some red cells, in particular by the disappearance of their central concavities, the globule taking on a more rounded appearance analogous to that of spherocytes or other globules or cells of diameter similar.

The diagnosis of various anemias is facilitated by the precise analysis of the sizes of the red blood cells and certain quantitative parameters such as Wintrobe indices of mean globular volume, mean hemoglobin level and mean globular hemoglobin concentration (globular value). Currently, these indications are typically obtained by means of a
Coulter which measures the number of red blood cells per cubic millimeter, the hemoglobin level and the average globular volume in a liquid circulation device. In addition to this data provided by the Coulter counter, it would be extremely useful to have a precise analysis of the globular species present in the sample divided into hematological categories: normocytes, macrocytes, target cells, microcytes, polkilocytes, hypochromic ovalocytes, spherocytes etc. A method and an apparatus making it possible to individually distribute globules in such categories and to obtain globular parameters on the red cells of each category are described in US Pat. No. 4,097,845 (James W. Bacus) entitled "Method and apparatus for the automatic classification of red blood cells ", which is formally reported as if it were reproduced here in its entirety.

The oldest and most common method of pre
trim microscopic slides for manual evaluation
the sub microscope includes the operation of spreading a
a certain amount of blood, carried by a thin glass layer on the surface of this blade using a second blade.

After the layer has dried, which only takes a short time,
soak the slide in a coloring agent, then observe the slide under a microscope and visually analyze the red blood cells. Or
Since this process is long, the physical effect exerted by the rubbing blade tends to deform many red blood cells. Typically,
under optimal conditions, only a fraction of the area su
of the mass is suitable for analysis. Because of these dice
training, this blade spreading technique is not suitable
for automatic red blood cell analysis.

We have developed, in particular for the analysis of
leukocytes, a routing technique used to distribute
on a slide of leukocytes in a single cell layer in view
of their subsequent automatic analysis. The device used essentially acts to rotate a blade and the blood it
door in a plane passing through the surface on which the
blood, excess blood being driven out by centrifugal effect of the over
face of the blade. By surface tension effects and / or
very, a layer of monocellular blood is retained on the slide.

For a leukocyte examination, the repaired globules are left to dry
sorted in a single-cell layer, then they are colored with a
Wright's dye or the like and are automatically analyzed as described in US Patent 3,883,852.

Although the drying of leukocytes does not cause
sensitive deformations from the point of view of leukocyte analysis,
we see that that of red cells causes indefinite alterations
Sirable, in particular the disappearance during drying of the concavities
many red blood cells. The reasons for these shape changes are not fully known, but these apparently result from the effect of surface tension, load and / or
drying.

The routing technique causes training on
the blade of the preferred single cell red cell layer, the hema
ties being separated, that is to say spaced, from each other.

If such a slide is immersed in a fixing or staining liquid before the cells are dry, the red cells are removed from the slide by washing, therefore it is desirable to fix the red cells after the formation of such a layer, but before drying .

 Consequently, the general object of the present invention is to propose a method making it possible to fix the cell morphology of cells carried by a slide after formation of the monocellular layer, but before drying of the cells on the slide, in order to preserve their appearance. characteristics for further analysis.

Other objects and advantages of the invention will emerge from the detailed description which will now be given with reference to the appended drawings, in which:
Figure 1 is a schematic top view, with magnification, of a microscopic slide carrying a monocellular layer of red blood cells, some of which have and others do not have a central concavity; Figure 2 is a side view corresponding to Figure 1;
Figure 3 shows in perspective, open, an apparatus according to the invention;
Figure 4 shows the same closed device.

In general, according to the process targeted by the invention, a certain quantity of blood sample is placed on a microscopic slide which is then caused to be driven by a centrifugal router. Routing drives blood away from the blade, subject to a single-cell layer. Before the sample has undergone drying normally sufficient to deform the red cells, a fixing agent is applied to the single-cell layer capable of preserving the forms of the red cells so that they do not undergo, during the subsequent drying, an alteration sufficient to make their central concavities disappear.

 FIG. 3 represents an apparatus for implementing the method described, comprising a centrifugal router 10 which comprises a casing 12 defining an interior chamber 14, open at the top, in which is disposed a rotary plate mounted on the end of a shaft 18. This plate receives on its flat upper surface a microscopic blade 20 in order to rotate it in a horizontal plane. The blade 20 is temporarily immobilized on the tray by suitable means 22 such as ears or the like, having on the surface of the tray the spacing desired to receive the blade. The shaft 18 is driven by motor means not shown, the speed and the routing time being controlled by conventional means well known to the technician. There are commercially available suitable routers, which can be of the kind described in US patents 3,853,092 and 3,906,890.

 According to one aspect of the invention, the open top of the router chamber can be covered by a cover 24, designed to rest on the casing 20 above the open top. In the example described, the cover 24 communicates, for example by a conduit 26, with a source of vapor of fixing agent, such as cartridge 28 containing the fixing agent, A shutter 30 interposed on the conduit 26 controls the flow passage of vapors from the source inside the chamber 14 of the router. In the example illustrated, the shutter 30 is a semi-circular cover 31 articulated in its center on an axis 33 fixed on the cover. The cover can be pivoted, to hide or unmask the orifice of the duct 26, by moving a gripping stud 35 which it carries in an arc of circle. For the vapors to flow towards the chamber, the cartridge is maintained 28 under pressure higher than that prevailing in the routing chamber, for example by means of a fan 32 mounted in the cartridge 28 or an air pump 34 sending air into the cartridge.

 As noted in the introduction, to prepare a blood sample for analysis, a certain quantity 36 of this sample is deposited on the blade 20, which is then mounted on the routing plate 16 disposed in the chamber 14. We do rotate this slide at the speed and for the time necessary to obtain a single-cell layer of red blood cells uniformly distributed over the surface of the slide. Currently, a constant routing speed of 4,000 to 10,000 rpm is maintained during the relatively short routing time, for example less than a second to 2.5 seconds, necessary for obtaining a satisfactory single-cell layer.

As described in the article by James W. Bacus "Erythro cyte Morphology and Centrifugal Spinner (Blood Film Preparations" published in The Journal of Histochemistry and Cytochemistry, 22: 7: 506-516, 1974, blood samples are preferably diluted to adjust their plasma viscosities in order to be able to obtain, by routing the sample for a constant time and at a constant speed, the single-cell layer of cells suitably separated and with well-marked central concavities.

For example, a whole blood sample can be diluted with a serum albumin solution with a relative viscosity of 1.3 to adjust the hematocrit of each sample to 18%. The relative viscosity is related to a value of 1.0 for H2O and a typical range of relative viscosities of blood plasma is from 1.2 to 1.8. Alternatively, a standard isotonic saline solution in a suitable ratio, for example 1: 1, can be used as the diluent for most blood. As the technician knows; if the routing time is too short, the globules are grouped instead of being isolated, and it is therefore impossible to classify them individually. If the routing time is too long, the globules undergo unacceptable deformations.

Instead of adjusting the viscosity of the plasma and providing a constant routing time, which is preferable, it is possible to use a routing device capable of adjusting the routing time. Such an apparatus, described in US Pat. No. 3,827,805, comprises a light beam which passes through the blade and the sample during routing. By measuring the degree to which the beam is dispersed by the globules, it is possible to stop routing when obtaining a predetermined distribution of the globules.

Another device for adjusting the routing time is described in US Pat. No. 3,906,890. This device allows the operator to determine the approximate concentration of the sample in blood cells and to adjust the routing time accordingly. The present invention can, but is not limited to, be implemented using one of these patented devices.

Advantageously, the single-cell layer of red blood cells carried by the blade is fixed and dried to the extent allowing its mechanical handling without breaking or displacement of the globules. Typically, a single-cell layer carried by a dry blade in 10 to 15 seconds after stopping the routing, this time varying with humidity and other conditions. So far, the drying of the single-cell layer causes, as noted in the introduction, annoying deformations of the red blood cells which interfere with identifying globules with central concavity and distort the identification of globular malformations. By way of example, the red cells 40 and 42 have, in FIG. 1, central concavities 44, which the lion fixes by means of a fixing agent arriving according to the arrows shown in FIG. 2 to prevent them from disappearing during subsequent drying of the globules.

If they have not been fixed before drying, the red blood cells flatten and take the forms indicated in dashed lines in 40a and 42a in FIG. 2. The red blood cells thus flattened in 42a and 40a, the central concavity of which is eliminated or strongly deformed, are difficult to distinguish from a true spherocyte 46 as shown in Figure 1, which has never presented a central concavity, by means of an automatic analysis device.

 However, it can be seen that, by the process described, the red cells can be adequately dried, without altering their globular morphology, by fixing them during or immediately after routing, when they are distributed in a single-cell layer with an adequate dispersion, but before drying has not progressed enough to alter the globular morphology to an unacceptable degree. It turns out that this fixation keeps the globules their physical geometry, so that the drying of the layer can continue without the globular deformations appearing according to the prior art. Preferably, the fixing is carried out once the layer has been formed and the routing stopped, so that it takes place before the drying is too pronounced. This result is obtained, according to the invention, by introducing into the routing chamber a fixing agent which is in the form of vapors, so that it has no undesirable effect on the blood sample. This fixation substantially ensures the preservation of the globular morphology.

 It has been found that fixing the globules operated before routing prevents the Ee globules from dispersing into the desired single-cell layer, so that they rather form clusters or strings during subsequent routing. It is also noted that the deposit, after routing, but before drying of a liquid fixing agent on the slide disturbs the distribution of globules, to the point that the blood sample is removed by washing the rotating blade.

A fixing agent which proves effective is formaldehyde, gaseous at normal room temperature. In the apparatus described, the cartridge 28 contains formalin (an aqueous solution of about 37-50% formaldehyde and about 15% methanol, by volume). The formaldehyde escapes from the solution and is channeled through the conduit 26 towards the chamber 14 of the router 10. As a variant, the blade 20 carrying the monocellular blood layer can be quickly removed and placed under an atmosphere of formaldehyde vapors. This variant is less suitable given the short time - about 5 seconds in which the transfer must be made to carry out the fixing before the drying has untoward effects. In addition, the "closed" circuit established by the The device shown is preferable because it keeps formaldehyde vapors trapped, toxic at a concentration exceeding 0.005 o / oo and dangerous at higher concentrations. An exposure of approximately five minutes of the single-cell layer to an atmosphere of formaldehyde vapors ensures the desired fixation of the red cells.

 During the routing, the sample is mainly driven from the blade on the inside of the router cover 12. This blood can contain harmful organisms, so that in certain routers, a liquid (usually water) is run off ) on the inside of the casing 12, during routing, to establish a hydraulic safety seal preventing these organisms from escaping from the chamber.

The projected blood is thus blocked by washing through a purge hole made in the bottom of the routing compartment. According to one embodiment of the invention, formalin is substituted for this runoff water. Thus, formaldehyde vapors are released in the router at the start of routing. According to this embodiment, the routing causes the formation of a single-cell layer within a fairly short time - of at least 0.5 seconds - so that, although formaldehyde vapors are initially present in the routing chamber, fixation takes place only after the globules have been dispersed in the form of a single-cell layer.

 Once the sample has been fixed and dried, the sample is removed from the routing or fixing chamber and an automatic analysis is carried out. It is found that red cells 40 and 42, fixed in the described manner, retain their characteristic central concavities 44 and other physical characteristics, so that the samples thus prepared make it possible to obtain, by analysis with the automatic analyzer, indications undistorted and precise on the morphology of the globules.

 Although the preferred fixing agent is formaldehyde, it should be possible to substitute it for other alcohols, methyl, ethyl or short chain, or to effect the fixing by other means such as heating, for example by microwave, or other physical or chemical method. The invention is therefore not limited to any particular fixing agent.

 In general, the arrangements described lend themselves to various modifications, without departing from the scope of the invention.

Claims (7)

 1. A method of preparing and fixing a specimen consisting of a single-cell layer of separate wet cells carried by a microscopic slide for subsequent automatic analysis, characterized in that: a certain quantity of the cells is placed on a support wet and the cells and the support are subjected to a routing, for a few seconds or less, in order to obtain on the support an adequate single-cell layer of separate cells and to project the excess cells out of the support, the routing is stopped then, before until the wet cells begin to dry out and change shape significantly, which occurs in 15 seconds or less under exposure to the ambient atmosphere, the wet cells are fixed with a fixing agent over a period of time significantly longer to preserve the shapes of the cells, and the cells fixed on said support are then dried to obtain a single-cell layer on the latter area of separate cells fixed and dried for subsequent self-analysis.  2. Method according to claim 2, characterized in that the wet cells are fixed by exposing them to formaldehyde vapors for a fixing time of at least five minutes.  3. Method according to claim 2, according to which the cells of the specimen comprise red blood cells, characterized in that the central concavities of the red blood cells are fixed before drying them and the other red blood cells are fixed to prevent it from n 'there appear artificial formations resembling, after drying, central concavities. 4. Method according to claim 1, characterized in that vapors of fixing agent are generated at a point external to a chamber in which said routing support is subjected and these vapors are sent into the routing chamber for fix the wet cells of the single-cell layer carried by the support. 5. Apparatus for implementing the method according to claim 1, characterized in that it comprises an envelope defining a routing chamber, a rotary plate disposed substantially horizontally in this chamber and capable of receiving a support on which wet cells are deposited, motor means capable of rotating said plate, means regulating the action time of these motor means so that the wet blood cells spread over the support in a substantially single-cell layer, and means ensuring the supply of vapors of fixing agent to these wet cells after their dispersion in a substantially single-cell layer on the support and before their drying accompanied by deformations. 6. Apparatus according to claim 5, characterized in that the envelope of the routing chamber has an inner surface and in that said means for supplying vapors of fixing agent comprise means causing a said stream to flow over said inner surface. liquid which establishes a security seal during the rotation of said plate, this liquid. evolving said vapors of fixing agent in the routing chamber.  7. Apparatus according to claim 5, characterized in that a channeling means brings vapors of fixing agent into said chamber, from an external source, to fix the wet cells while they are still in the routing chamber.