Method for the preparation of blood samples for automated analysis

A method for preparing blood samples containing red blood cells for automated analysis wherein the blood sample on a slide is spun to create a monolayer of randomly distributed red blood cells. To inhibit cell morphology distortions from occurring during drying, the morphologies of the cells contained in the monolayer are preserved by a fixing agent after monolayer preparation but prior to drying when such distortions would otherwise develop. The method and apparatus are particularly useful for fixing red blood cells to prevent loss of or deformation of a central pallor which would be detrimental to a subsequent automated analysis.

This invention relates to a method of, and an apparatus for, the 
preparation of blood cell specimens for diagnostic analysis. More 
specifically, the invention is particularly directed to the fixing or 
preservation of the morphology of cells for later automated analysis. 
The present invention is particularly directed to fixing cells during a 
precise and critical step of sample preparation, such that their 
morphology is preserved for later analysis. Thus, the development of 
distortions encountered by other known methods of sample preparation 
having cells on a slide is reduced to a minimum. Although the present 
invention is not to be construed as limited to any particular kind of cell 
or sample, it will be described in connection with the preparation of red 
blood cells for automatic analysis on a rapid basis. One distortion noted 
heretofore in known methods of preparation of microscopic slides was that 
of the alteration of morphology of certain red blood cells, particularly 
the loss by certain cells of their central pallor, i.e., a thin central 
indented region in a cell, with the cell becoming more rounded and 
resembling spherocytes or other cells of a similar diameter or shape. 
The diagnosis of various kinds of anemia is enhanced by accurate analysis 
of the sizes of the red blood cells and the quantification of red cell 
parameters, such as the Wintrobe indices of mean cell volume, mean cell 
hemoglobin and mean cell hemoglobin concentration. At the present time, 
such information is typically acquired by the use of a Coulter Counter 
which measures the number of red blood cells per cubic millimeter, the 
hemoglobin concentration, and the mean cell volume in a liquid flow 
system. In addition to this information provided by the Coulter Counter, 
it would be most helpful to have an accurate analysis of the kinds of 
cells present in the sample in accordance with established hematological 
classifications, such as normocytes, macrocytes, target cells, microcytes, 
spiculed cells, hypochromic elongated cells, spherocytes, etc. A method of 
and apparatus for classifying individual cells into such classifications, 
i.e., subpopulations, and providing red blood cell parameters for each of 
the subpopulations, is disclosed in co-pending application Ser. No. 
737,531 of James W. Bacus, entitled "A Method of and an Apparatus for 
Automatic Classification of Red Blood Cells", filed on even date, this 
application being incorporated by reference as if fully reproduced herein. 
The oldest and most common method of preparing microscopic slides for a 
manual evaluation under a microscope involves the wedge-slide technique in 
which a quantity of blood on a glass slide is wiped by a second slide 
along the surface of the first slide to produce a thin blood layer on the 
first slide. After the layer dries, which requires only a short time, the 
slide is dipped into a staining agent and then the operator views the 
slide under a microscope and visually analyzes the red cell population. In 
addition to being time consuming, the physical action of the wiping slide 
tends to distort the morphology of many of the cells. Typically, under the 
best of conditions, only a fraction of the surface area of the slide is 
suitable for analysis. This distortion renders this wedge technique 
unsuitable for automated red blood cell analysis. 
There has been developed, particularly for white blood cell analysis, a 
spinning technique for the formation of a monolayer of cells on a slide 
for later automated analysis of the white blood cells. Essentially, such 
an apparatus spins a slide and the blood placed thereon in the plane of 
the surface on which the blood rests with the excess blood being hurled 
outwardly from the slide surface. Surface tension and/or other forces 
retain a monolayer of blood upon the slide. For white blood cell work, the 
cells in the monolayer are allowed to dry and then are stained with a 
Wright's stain (or other stain) and analyzed automatically such as 
disclosed in U.S. Pat. No. 3,883,852. 
While the drying of the white blood cells has not produced a substantial 
distortion from a white blood cell analysis standpoint, it has been found 
that the drying of the red blood cells produces undesirable types of 
distortions, particularly a loss of central pallor for many of the red 
blood cells as they dry. It is not entirely clear what causes these shape 
changes, but they apparently are caused by surface tension, charges and/or 
drying effects. 
The spinning technique causes the preferred monolayer of red blood cells to 
be formed on the slide with the red blood cells separated, that is, spaced 
from each other. Should such a slide be dipped into a fixing or staining 
liquid before the cells are dry, the red blood cells would be washed from 
the slide. If the red blood cells are fixed in solution, prior to 
deposition on the slide, the fixation causes them to cluster together such 
that a monolayer of separated cells is not obtained. Thus, it is desirable 
to fix the cells during the period after monolayer formation, but prior to 
drying. 
Accordingly, a general object of the invention is to provide a method for 
fixing cell morphology of cells on a slide after the monolayer formation, 
but prior to the drying of the cells on the slide to preserve their 
identifying features for later analysis.

Generally, in accordance with the method of the present invention, a 
quantity of blood sample is placed upon a microscope slide which is then 
spun by a centrifugal spinner. The spinning action of the slide throws all 
but a monolayer of the blood from the slide. Before the sample has dried 
to the extent that red cells would normally be distorted by such drying, a 
suitable fixing agent is applied to the monolayer to preserve the shapes 
of the red cells so that further drying does not adversely change the cell 
morphology to the extent of losing the central pallor configurations. 
Referring to FIG. 3, an apparatus for practicing the disclosed method 
comprises a centrifugal spinner 10 including a housing 12 defining a 
top-opening interior chamber 14 within which there is disposed a platen 16 
rotatably mounted on the end of a shaft 18. The platen receives a slide 20 
on the upper horizontal flat surface of the platen for spinning of the 
slide in a horizontal plane. The slide 20 is releasably held in position 
on the platen by appropriate means 22 such as lugs or the like that are 
spaced apart on the platen surface to receive the slide. The shaft 18 is 
driven by motor means (not shown) with the rate and duration of spinning 
being controlled by conventional means well known in the art. Suitable 
spinners are commercially available, and may be of the kind shown in U.S. 
Pat. Nos. 3,853,092 and 3,906,890. 
In accordance with one embodiment of the disclosed invention, the top 
opening of the chamber of the spinner is closable as by a covering hood 24 
adapted to rest on the housing 12 in position over the top opening. In the 
depicted embodiment, the hood 24 is connected in communication, as by a 
conduit 26, with a source of vaporous fixing agent, such as a canister 28 
containing the fixing agent. A valve means 30 interposed in the conduit 26 
intermediate its opposite ends provides control over the flow of vapor 
between the source and the interior chamber 14 of the spinner. Herein, the 
valve 30 comprises a semi-circular valve plate 31 pivoted at the center by 
a pin 33 fixed on the cover hood. The valve plate is pivoted to cover or 
uncover the discharge orifice of the conduit 26 when an upstanding handle 
pin 35 is gripped and swung in an arc. To the end of urging the flow of 
vapor to the chamber, the pressure within the canister is maintained at a 
higher level than the pressure within the spinner chamber, such as by a 
fan 32 mounted within the canister 28 or by an air pump 34 supplying air 
to the canister. 
As noted above, in preparing a blood sample for analysis, a quantity of the 
blood sample 36 is placed upon the slide 20 which is then mounted upon the 
spinner platen 16 disposed within the chamber 14. The slide is spun at a 
rate and for a period of time necessary to develop an evenly distributed 
monolayer of red blood cells upon the surface of the slide. Commonly, the 
spinning rate is held constant, at a rate between 4,000 and 10,000 rpm 
with the actual period of spinning being relatively short, e.g., less than 
one second to 2.5 seconds, as is required to develop a satisfactory 
monolayer. 
As disclosed in an article by James W. Bacus, "Erythrocyte Morphology and 
Centrifugal `Spinner` Blood Film Preparations", The Journal of 
Histochemistry and Cytochemistry, 22:7:506-516, 1974, the blood samples 
are preferably diluted to adjust their plasma viscosities such that the 
sample may be spun for constant spin time and at a constant speed to 
provide the monolayer of cells having good separation and central pallor 
development. By way of example, a thoroughly mixed sample of whole blood 
was diluted with a serum albumin solution of 1.3 relative viscosity to 
adjust the hematocrit of each sample to 18%. The viscosity was relative to 
H.sub.2 O as 1.0 and a typical range of relative viscosities for blood 
plasma is 1.2-1.8. Alternatively, a standard isotonic saline solution in a 
convenient ratio, e.g., 1:1, may be used as a diluent for most bloods. As 
is recognized in the art, if the spinning period is too short, the cells 
are clustered together and thus impossible to isolate and classify 
individually. If the spinning period is too long, there is an undue 
distortion of the cell shapes. 
Rather than adjusting the plasma viscosity and using a constant spin time, 
as preferred, spin apparatus may be used which controls the spining 
period. One of these is disclosed in U.S. Pat. No. 3,827,805 and employs a 
light beam which passes through the slide and sample during spinning. By 
measuring the degree of scattering of the beam caused by the blood cells, 
spinning may be stopped when a predetermined cell distribution is 
achieved. Another apparatus for controlling the spinning period is 
disclosed in U.S. Pat. No. 3,906,890. Using this apparatus, the operator 
determines the approximate blood cell concentration of the sample and sets 
the spinning time in accordance therewith. The present invention may be 
used with either of these patented apparatuses, but is not limited to 
either such apparatus. 
Desirably, the monolayer of red blood cells on the slide is fixed and dried 
to the degree that permits mechanical handling of the slide without 
disruption or dislocation of the cells. Typically, a monolayer of blood on 
a slide air dries within 10 to 15 seconds after spinning is stopped, the 
time varying with humidity and other conditions. Heretofore, drying of the 
monolayer, as noted above, resulted in deleterious distortion of the red 
blood cells with resultant lack of identification of cells with central 
pallor and false identification of the misshapen cells. By way of example, 
red blood cells 40 and 42 are illustrated in solid lines in FIG. 1 as 
having a central pallor area 44, which is fixed by a fixing agent 
indicated by arrows in FIG. 2, to fix the central pallor against the loss 
thereof during a subsequent drying of the cells. Without fixing the cells 
40 and 42 prior to drying, the cells flatten and assume the flattened 
shapes 40a and 42a, as shown in dotted lines in FIG. 2. The flatten cells 
42a and 40a having a lost or severely distorted central pallor region are 
found to be difficult to distinguish from a true spherocyte cell 46, which 
never had a central pallor, as seen in FIG. 1, by automated analysis 
equipment. 
In accordance with the disclosed method, it has been found that the red 
blood cells can be dried adequately without alteration of the cell 
morphology by fixing the red blood cells during the course of or 
immediately after the spinning, when the cells have attained a monolayer 
status with proper dispersion of the cells, but prior to the time when the 
drying process has progressed to the extent that the cell morphology is 
unacceptably altered. Such fixation has been found to set the physical 
geometry of the cells whereby the drying of the monolayer can proceed 
without the cell distortion noted in the prior art. In a preferred 
embodiment, the fixation step is performed when the monolayer has been 
established and the spinning force has been removed, so that the fixation 
occurs before too much drying has occurred. This is accomplished in 
accordance with the present disclosure by introducing into the chamber in 
which the slide is spun a fixing agent that is in the form of a vapor, so 
that its presence causes no ill effect upon the blood sample. This 
fixation step substantially sets the cell morphology. 
Fixing of the cells prior to spinning has been found to prevent the 
dispersion of the cells in the desired monolayer, resulting rather in 
clumping or stringing of the cells when they subsequently are spun. Also, 
the introduction of a liquid fixing agent onto the slide, after spinning, 
but before drying, has been found to disrupt the cell dispersion, even to 
the point of washing the blood sample from the spinning slide. 
One fixing agent which has proven effective is formaldehye, a gas at normal 
room temperatures. In the depicted apparatus, formalin (an aqueous 
solution of between about 37% and 50% formaldehyde and about 15% methanol, 
by volume), is stored in the canister 28. The formaldehyde escapes from 
the solution and is conveyed to the chamber 14 of the spinner 10 by the 
conduit 26. Alternatively, the slide 20 with the monolayer of blood 
thereon can be quickly removed from the spinner chamber and placed in an 
atmosphere of vaporous formaldehyde. This latter step is less desirable in 
view of the relatively short time, i.e., about 5 seconds, available within 
which the transfer must be made if the fixation is to take effect before 
deleterious drying occurs. Further, the "closed" system in the depicted 
embodiment is preferred as an aid in controlling the vaporous formaldehyde 
which is toxic at levels greater than 5 ppm and creates a risk at 
concentrated levels. Exposure of the monolayer for about five minutes to 
an atmosphere of vaporous formaldehyde provides the desired fixation of 
the red blood cells. 
When the blood sample is spun, the vast majority of the sample is spun from 
the slide to the interior walls of the spinner housing 12. This blood may 
contain a variety of harmful organisms, so that in some spinners, a liquid 
(usually water) is cascaded over the interior walls of the housing 12 
during spinning to serve as a safety seal against escape of such organisms 
from the chamber. The flying blood is thus washed away through a drain in 
the bottom of the spinning compartment. One method of practicing the 
present invention includes replacing this cascading water with formalin. 
Thus, formaldehyde vapors are produced in the spinner from the time 
spinning commences. In this method, the spinning and establishment of a 
monolayer occurs sufficiently rapidly, i.e., within about 0.5 seconds, so 
that even though the vaporous formaldehyde is initially present in the 
spinning chamber, fixation does not occur until after the cells have been 
dispersed into the monolayer. 
After the sample has been fixed and dried, the slide 20 is removed from the 
spinner or fixing chamber and analyzed by automated means. Red blood cells 
40 and 42, fixed in the manner described herein, have been found to retain 
their characteristic central pallors 44 and other physical characteristics 
such that the samples so prepared provide a true and accurate indication 
of the morphology of the cells when analyzed using automated analyzers. 
While a preferred fixing agent of formaldehyde has been used effectively, 
it is expected that other, methyl, ethyl or short-chain alcohols may be 
used in lieu of formaldehyde, or that other fixations, such as heating, 
for example, by microwaves, or other physical or chemical means may be 
used. Therefore, the present invention is not to be construed as limited 
to any particular fixing agent. 
While a preferred embodiment has been shown and described, it will be 
understood that there is no intent to limit the invention by such 
disclosure, but rather, it is intended to cover all modifications and 
alternative constructions falling within the spirit and scope of the 
invention as defined in the appended claims.