U.S. patent application number 11/887259 was filed with the patent office on 2009-03-12 for optical device for blood analysis, analysis apparatus equipped with such a device.
This patent application is currently assigned to C2 DIAGNOSTICS. Invention is credited to Serge Champseix, Laurent Damonneville, Olivier Magnin.
Application Number | 20090068726 11/887259 |
Document ID | / |
Family ID | 35033677 |
Filed Date | 2009-03-12 |
United States Patent
Application |
20090068726 |
Kind Code |
A1 |
Magnin; Olivier ; et
al. |
March 12, 2009 |
Optical Device for Blood Analysis, Analysis Apparatus Equipped With
Such a Device
Abstract
Optical device (200) for blood analysis is in particular
designed for the counting and differentiation of leucocytes in an
automatic blood analysis apparatus and includes a light source
(201) of the electroluminescent diode type in order to illuminate a
blood sample (311) circulating in an optical tank.
Inventors: |
Magnin; Olivier;
(Montpellier, FR) ; Damonneville; Laurent;
(Castelnau le lez, FR) ; Champseix; Serge;
(Tarnac, FR) |
Correspondence
Address: |
YOUNG & THOMPSON
209 Madison Street, Suite 500
ALEXANDRIA
VA
22314
US
|
Assignee: |
C2 DIAGNOSTICS
MONTPELLIER
FR
|
Family ID: |
35033677 |
Appl. No.: |
11/887259 |
Filed: |
March 22, 2006 |
PCT Filed: |
March 22, 2006 |
PCT NO: |
PCT/FR2006/000620 |
371 Date: |
September 27, 2007 |
Current U.S.
Class: |
435/288.7 |
Current CPC
Class: |
G01N 15/1456 20130101;
G01N 2015/1486 20130101; G01N 15/1434 20130101 |
Class at
Publication: |
435/288.7 |
International
Class: |
C12M 1/00 20060101
C12M001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2005 |
FR |
0503116 |
Claims
1. Optical device (200) for the counting and differentiation of
leucocytes in an automatic blood analysis apparatus (20),
characterized in that it comprises a light source (201) of the
electroluminescent diode type in order to illuminate a blood sample
circulating in an optical tank (300) along an injection axis
(X300).
2. Device according to claim 1, characterized in that the diode
(201) emits light the wave length of which is less than 600
nanometres.
3. Device according to claim 2, characterized in that the diode
emits light the wave length of which is less than 500
nanometres.
4. Device according to claim 1, characterized in that it emits a
source light beam (211) with a width comprised between 5 and 200
microns close to the injection axis.
5. Device according to claim 4, characterized in that it emits a
light beam with a width comprised between 90 and 120 microns.
6. Device according to claim 1, characterized in that the light
beam is emitted approximately in the direction of the tank,
approximately transversely to the direction of flow of the
sample.
7. Device according to claim 4, characterized in that it comprises
a rotatably-mounted transparent slide (220) arranged between the
diode and the tank in order to be passed through by the source
light beam (211) between two opposing surfaces.
8. Device according to claim 7, characterized in that the
transparent slide is rotatably mounted about an axis (X220)
approximately parallel to the movement of the blood sample in the
tank.
9. Device according to claim 1, characterized in that it comprises,
beyond the optical tank, means for separation (205) by Fresnel
losses for a resulting light beam (212) originating from the source
light beam, separating said beam into an axially-resulting beam
(213) and at least one beam resulting from Fresnel loss (214)
constituted by Fresnel losses while passing the separation means
(205).
10. Device according to claim 9, characterized in that the
separation means comprise at least one separation surface which is
a surface of a transparent separation material, the
axially-resulting beam having passed through the transparent
material and the beam resulting from Fresnel loss having been
reflected by the separation surface, said surface being slanted
with respect to the resulting light beam, beyond the tank.
11. Device according to claim 9, characterized in that it also
comprises an apparatus (222) for measurement of the light of the
axially-resulting beam and at least one other apparatus (223) for
measurement of the light of the at least one beam resulting from
loss.
12. Device according to claim 11, characterized in that the
measurement apparatuses (222, 223) comprise means for one
measurement from a measurement of fluorescence.
13. Device according to claim 11, characterized in that the
measurement apparatuses (222, 223) comprise means for a measurement
of the light losses in the axis.
14. Device according to claim 11, characterized in that the
measurement apparatuses (222, 223) comprise means for a measurement
of the diffraction close to the axis.
15. Device according to claim 1, characterized in that it also
comprises means for measurement of the diffraction of the light
beam at wide angles by the sample in the tank.
16. Device according to claim 1, characterized in that it
comprises, on the path of the beam before the tank, at least one
diaphragm (202) to block spurious light.
17. Haematology apparatus (20), in particular an automatic blood
analysis apparatus, characterized in that it comprises a device
(200) according to claim 1.
18. Device according to claim 10, characterized in that it also
comprises an apparatus (222) for measurement of the light of the
axially-resulting beam and at least one other apparatus (223) for
measurement of the light of the at least one beam resulting from
loss.
19. Device according to claim 12, characterized in that the
measurement apparatuses (222, 223) comprise means for a measurement
of the light losses in the axis.
20. Device according to claim 12, characterized in that the
measurement apparatuses (222, 223) comprise means for a measurement
of the diffraction close to the axis.
Description
[0001] The present invention relates to an optical device for the
counting and differentiation of leucocytes in an automatic blood
analysis apparatus, and an analysis apparatus equipped with such a
device.
[0002] Analysis of a blood sample generally seeks to determine:
[0003] the total number of leucocytes; [0004] more specifically,
the number of leucocytes by sub-populations (basophils,
eosinophils, neutrophils, monocytes and lymphocytes); [0005] the
number of erythrocytes and platelets; and [0006] the haemoglobin
level.
[0007] Several analysis techniques are known, in particular:
[0008] assay of the haemoglobin is carried out after lysis of the
erythrocytes, i.e. the destruction of the membrane of the cells of
erythrocytes, and by measurement by spectrophotometry of the
haemoglobin released in the medium; the assay of the haemoglobin
also requires the stabilization of the haemoglobin in a complexed
form (oxyhemoglobin or cyanmethemoglobin) in order to measure the
absorbance of a single compound at the appropriate wave length.
[0009] total leucocyte count is carried out on the blood sample by
resistivity with specific lysis of the erythrocytes and protection
of the leukocytes.
[0010] differentiation of the leucocytes and the counting thereof
by sub-population is carried out: [0011] either by resistivity
volumetric measurement after specific lysis of the erythrocytes,
protection of the leucocytes and adjustment of the pH; this however
does not allow differentiation of all the sub-populations in a
single analysis; [0012] or by optical way, in particular by flow
cytometry; after specific lysis of the erythrocytes and protection
of the leucocytes, by measuring different parameters (in particular
diffraction, fluorescence, absorbance), on a flow of leucocytes in
the axis of the narrow, medium and wide angles and optionally after
addition of a labelling agent (for example chlorazol black, or a
DNA or RNA labelling dye, or a fluorescent dye) and by measuring at
different wave lengths; this technique allows differentiation of
the sub-populations of leucocytes.
[0013] the erythrocyte and platelet count is carried out on a
diluted sample without the addition of specific reagent by
resistivity measurement.
[0014] Numerous automatic blood cell analyzers exist which use
these techniques in order to obtain a blood sample analysis which
is as complete as possible.
[0015] In these automatic apparatuses, two different analysis
circuits traditionally coexist: [0016] a first circuit designed to
measure the haemoglobin and/or the total leucocyte count; and
[0017] a second circuit designed to carry out on the blood sample
the differentiation and/or a leucocyte count by flow cytometry.
[0018] Each circuit is characterized by a dilution rate of the
blood sample suited to the measurement means used, the addition of
one or more reagents and appropriate means for implementation and
measurement.
[0019] Thus, for the measurement of haemoglobin and the counting of
the leucocytes, the circuit typically comprises a so-called
counting tank in which the blood sample is diluted, a reagent in
particular comprising the lysis compound of the erythrocytes, the
stabilisation compound of the complex formed from the haemoglobin
and the leucoprotective compound is added to it, and the following
are measured directly in this cell: haemoglobin by
spectrophotometry and the number of leucocytes by resistivity. The
dilution rate is chosen so that the analysis solution is perfectly
homogeneous and so that the detection apparatus is not saturated.
This dilution rate is comprised between 1/100.sup.th and
1/500.sup.th, generally between 1/160.sup.th and 1/180.sup.th.
[0020] For leucocytic differentiation by flow cytometry, the
circuit uses a tank for dilution of the blood sample to which one
or more reagents containing an erythrocyte lysis agent, optionally
a differentiation agent (for example a DNA or RNA leucocyte
fluorescent dye) are added, then a fraction of this solution is
taken in order to inject it into a flow-through optical tank of a
flow cytometer. The dilution rate used here is less than
1/100.sup.th, allowing an optimal analysis time to be obtained with
the cytometers currently available on the market (of the hydrofocus
type).
[0021] Thus, conventionally, at least two different reagents must
usually be used for the two analysis circuits and two different
dilutions of the blood sample are carried out in these two analysis
circuits.
[0022] The main objectives of manufacturers are to simplify the
existing automatic apparatuses by reducing the number of components
and reagents, allowing reduction of the production and maintenance
costs and the size of the automatic apparatuses, without however
reducing the time of a complete blood sample analysis.
[0023] The present invention in particular aims to achieve these
objectives.
[0024] Document WO 2004/003517 for this purpose proposes a method
and equipment in which the two analysis circuits have means in
common. The principle is to carry out a first dilution of the blood
sample in a single dilution tank and to successively transfer
fractions of selected volumes of this dilution to a measuring or
counting unit, in order each time to measure or count different
elements contained in the blood sample. In order to carry out a
complete analysis, namely counting the erythrocytes and platelets,
counting the leucocytes, measurement of the haemoglobin and
leucocytic differentiation, the document describes the following
solution: using a first transfer to count the erythrocytes and
platelets, adding a lysis agent to the dilution tank, then carrying
out a second transfer to count the leucocytes, carrying out a third
transfer of lyzed dilution solution to measure the haemoglobin
level, adding a leucocytic differentiation reagent, and carrying
out a fourth transfer to realize the leucocytic differentiation in
the measuring unit.
[0025] This principle may allow the use of a single so-called
dilution tank, but it does not allow a saving of analysis time
because the measurements or counting are carried out successively
after each transfer of a fraction of the dilution. Moreover, it
requires perfect control of the successive volumes of reagents and
diluents transferred to the measuring unit. Moreover it also
requires the use of several syringes and lysis reagents.
[0026] The objective of the present invention is also to overcome
such drawbacks.
[0027] According to a first object, the present invention relates
to a method for the automatic analysis of a blood sample as well as
a mono-reagent and an apparatus for implementing this method.
[0028] The method according to the invention is characterized in
that:
[0029] an analysis solution containing said blood sample, a
diluent, and: [0030] at least one compound to lyze the
erythrocytes; [0031] at least one compound to protect the
leucocytes; and [0032] at least one compound to stabilize the
haemoglobin in the form of a chromogenic complex; is formed in a
single dilution and analysis tank,
[0033] the haemoglobin level is measured in this analysis solution
by spectrophotometry in said tank after the lysis of the
erythrocytes; and
[0034] an appropriate quantity of this analysis solution is taken
from said tank on which a leucocytic differentiation is carried out
by an optical means.
[0035] The counting of the leucocytes can be carried out jointly in
the analysis tank and/or with the optical means.
[0036] The counting of the erythrocytes and optionally of the
platelets can be carried out for example in a previous stage of the
method on a sample carried out in the single dilution and analysis
tank.
[0037] Thus, the present invention is based on the concept of a
single analysis solution used as is for the two types of analyses
which were usually carried out in two separate circuits, namely on
the one hand the measurement of the haemoglobin and optionally the
counting of the leucocytes and, on the other hand, the leucocytic
differentiation by optical means, said analysis solution combining
the "reagent" compounds capable of carrying out at least these
analyses by virtue of their nature and their quantity. The reagent
compounds introduced are chosen to be chemically compatible with
each other and in quantities suited to the targeted analyses. They
can be chosen from the compounds typically used in the prior art.
It is also possible to use a commercial formulation which is
conventionally used to carry out a leucocytic differentiation, i.e.
containing the compound for lyzing the erythrocytes and the
leucoprotective compound, and to add to it the third reagent
compound intended to stabilize the haemoglobin in the form of a
chromogenic complex.
[0038] Due to this single analysis solution, the present invention
in particular has the following advantages: [0039] the automatic
apparatus can comprise a single tank for preparation of the
analysis solution, [0040] the measurement of the haemoglobin can be
carried out directly in this tank, and also the global counting of
the leucocytes by resistivity measurement of the analysis solution;
[0041] it is possible to use a mono-reagent combining all the
"reagent" compounds required for the measurement of the haemoglobin
and for the leucocytic differentiation by optical means; this in
particular allows simplification of the hydraulic circuits as will
be seen below; [0042] a mono-dilution of the blood sample can be
carried out directly in the single dilution and analysis tank, with
a dilution rate determined as a function of the measurement and
detection means used. The mono-reagent can serve as a diluent for
carrying out this mono-dilution. Preferably, a dilution rate will
be chosen comprised between 1/100.sup.th and 1/500.sup.th,
corresponding to the dilution rate required for a measurement of
the haemoglobin level, preferably also a rate of approximately
1/175.sup.th ( 1/173.sup.rd in the embodiment given below).
[0043] With the possibility of using a mono-dilution and a
mono-reagent, it is therefore possible, thanks to this first aspect
of the invention, to greatly simplify the analysis equipment while
still providing a complete analysis of the blood sample.
[0044] Means for optical measurement allowing an analysis of the
leucocytes (counting and differentiation by sub-populations) at a
dilution rate greater than 1/100.sup.th are also proposed according
to the invention and are defined and described below.
[0045] The present invention also proposes a lysis mono-reagent for
the implementation of the method according to the invention,
characterized in that it comprises: [0046] at least one compound to
lyze the erythrocytes; [0047] at least one compound to protect the
leucocytes; and [0048] at least one compound to stabilize the
haemoglobin in the form of a chromogenic complex.
[0049] Such a mono-reagent allows measurement by spectrophotometry
of the hemoglobin concentration of a blood sample and a leucocytic
differentiation by optical means. It also allows the resistive
and/or optical counting of the leucocytes. Preferably it is chosen
so as to allow the differentiation of at least 5 sub-populations.
Preferably it is chosen so that it does not contain cyanides.
[0050] According to the invention, the compound to lyze the
erythrocytes is preferably constituted by at least one cationic
surfactant. In a preferential manner which is known per se, it is
chosen to form an oxyhemoglobin complex (as it is non-toxic
compared to a cyanmethemoglobin complex which involves cyanide
ions). The cationic surfactant is therefore also chosen such that
it oxidizes the released haemoglobin so as to form only an
oxyhemoglobin complex. The quantity of cationic surfactant is
therefore chosen so as to efficiently haemolyse the erythrocytes
and oxidize the haemoglobin released. It is preferably chosen from:
[0051] the quaternary ammonium salts, preferably
alkyltrimethylammonium salts and still more particularly cetyl-,
dodecyl-, tetradecyl- and hexadecyltrimethylammonium bromides and
chlorides; [0052] pyridinium salts; [0053] long-chain ethoxylated
amines; and [0054] alkyl sulphates (SDS).
[0055] The leucoprotective compound according to the invention is a
compound which delays or prevents the destruction of the
leucocytes. Preferably it is a non-ionic or amphoteric surfactant
preferably chosen from: [0056] ethoxylated alcohols, in particular
2-phenoxyethanol, polyoxyethylenealkylphenylethers, such as the
commercial products IPEGAL990.RTM., TERGITOL NP9.RTM., TRITON.RTM.
X100 or X114, plurafac.RTM. A38 or Brij35.RTM.); [0057] betaines
and sulphobetaines of quaternary ammoniums in particular
lauramidopropyl betaine (LAB), and
dodecyldimethyl-3-ammonio-1-propanesulphonate (DDAPS) or
tetradecyldimethyl-3-ammonio-1-propanesulphonate (TDAPS);
[0058] tertiary amine oxides, such as
N,N-dimethyllaurylamine-N-oxide (LDAO) or
3-[(cholamidopropyl)-dimethylamino-]-1-propane sulphonate (CHAPS or
CHAPSO); [0059] the glycosidic type compounds and more particularly
a triterpene saponin; [0060] the glucidic type compounds (mannitol,
D-glucose, trehalose, dextran sulphate).
[0061] The compound which stabilizes the haemoglobin in the form of
a chromogenic complex is preferably chosen from:
[0062] mono or polydentate chelates presenting ligand atoms
(non-binding pairs: O, N, S, and carboxy COO-groups etc.) in
particular: [0063] ethylene diamine tetraacetic acid (EDTA) or
ethylene glycol-bis-(3-aminoethylether)N--N'-tetraacetic acid
(EGTA) and in particular their sodium or dipotassium salts; [0064]
potassium oxalate K.sub.2O.sub.xO.sub.x.dbd.C.sub.2O.sub.4.sup.2-;
[0065] hydroxylamine salts (preferably hydrochlorites); and [0066]
organic acids (in particular formic or acetic).
[0067] the aromatic compounds (mono or polydentate chelates)
comprising ligand atoms (having non-binding pairs: O, N, S etc.),
in particular: [0068] Tiron.RTM. [0069] 8-hydroxyquinoline and its
derivatives; [0070] pyridine or bipyridine and their derivatives;
[0071] 1,10-phenanthroline and its derivatives; [0072] the phenolic
compounds (mono or bis and their derivatives); [0073] pyrazole
and/or the pyrazolones and their derivatives; [0074] imidazole and
its derivatives; [0075] sulphosalicylic acid; and
[0076] saponins, tertiary amine oxides, betaines and sulphobetaines
of quaternary ammoniums (such as DDAPS, TDAPS, LAB).
[0077] In addition to the three compounds defined according to the
invention, it is possible to add to the (mono)-reagent(s):
[0078] at least one dye (or mixture) specifically labelling certain
leucocytes and more particularly eosinophils (or basophils), in
order to allow the distinguishing of at least the 5 main leucocyte
sub-populations, chosen from: [0079] cyanines; [0080] Oxazine 750;
[0081] Wright and Romanowsky reagents; [0082] DAPI; [0083]
Clorazole black E; [0084] Toluidine blue; [0085] Astra Blue; [0086]
thiazole orange G. or blue; [0087] other fluorescent reagents.
[0088] at least one fixing agent allowing stiffening of the
membrane of the leucocytes which is preferably an aldehyde and more
particularly glutaraldehyde or formaldehyde;
[0089] at least one wetting agent in order to optimize the fluidics
and prevent the formation of bubbles which also act as solubilizers
of the debris, chosen from: [0090] alcohols (methanol ethanol or
propan-2-ol); [0091] glycols (ethylene or propylene glycol); [0092]
ethoxylated glycols (particularly Triton X100.RTM. or Brij35.RTM.);
[0093] glycosidic compounds TWEEN80.RTM. or TWEEN20.RTM.; the
concentration of the fixing agent and of the solubilizer being
strictly limited, as an excess can prevent the lysis of the
erythrocytes and modify the optical properties of the leucocytes;
and
[0094] a buffer system for setting the pH between 5.0 and 10.0 and
preferably between 6.0 and 8.0 and optimally close to neutrality
(7.0.+-.0.4). The choice of such a pH aims to respect the native
conditions of the cells. Moreover this pH allows a better
dissolution of the constituents used according to the invention.
Said buffer is constituted by a pair of salts (inorganic or
organic) adjusted to the above-mentioned pH by hydrochloric acid or
soda (4-6N), chosen from: [0095] sodium or potassium dihydrogen
phosphate/hydrogen phsophate
H.sub.2PO.sub.4.sup.-/HPO.sub.4.sup.2-; [0096] sodium hydrogen
carbonate/carbonate NaHCO.sub.3/Na.sub.2CO.sub.3 [0097] a citric
acid/sodium citrate (III) buffer [0098] TRIS-HCl [0099]
triethanolamine (TEA) [0100] imidazole
[0101] an acid chosen from: [0102] the organic acids: phthalic,
sulphosalicylic or formic, which also contribute to the formation
and stabilization of the chromogenic complex of the haemoglobin);
and [0103] the mineral acids: HCl, H.sub.3PO.sub.4 etc.
[0104] a background salt ensuring a conductivity of the order of 10
to 50 ms/cm required for the resistivity measurement and an
osmolarity of the order of 120 to 500 mOsm and preferably close to
isotonicity (290.+-.5 mOsm), chosen from: [0105] sodium chloride
NaCl; [0106] potassium chloride KCl; [0107] magnesium chloride
MgCl.sub.2; [0108] calcium chloride CaCl.sub.2; [0109] anhydrous
sodium sulphate Na.sub.2SO.sub.4;
[0110] this background salt being able to be comprised in the
buffer system;
[0111] at least one preservative, having antioxidant, and/or
antibiotic properties chosen from: [0112] 2-phenoxyethanol; [0113]
parabens; [0114] BHT; [0115] isothiazolones (Proclin.RTM. 150 or
300); [0116] imidazole or urea derivatives; [0117] antibiotics;
[0118] a natural antibiotic cellular penetration compound
(ionophore) which also facilitates the penetration of the dye or
dyes chosen from: [0119] ionophore I for NH.sub.4.sup.+ (nonatine);
[0120] ionophore III for Ca.sup.2+ (calcimycine); [0121] ionophore
for Cl.sup.-; [0122] ionophore I for K.sup.+ (valinomycine).
[0123] The constituents according to the invention are summarized
in the table below as well as ranges of appropriate
concentrations.
TABLE-US-00001 Constituent Quantity Cationic surfactant (lysis
agent) 0.1-50 g/L Leucoprotective surfactant 0.1-20 g/L Chelate of
the haemoglobin 0.0001-10 g/L complex Dye 0.01-1 g/L Fixing agent
0.01-2% w/v Wetting agent 0-50% v/v Buffer 0-6 g/L Background salt
1-50 g/L Acid Appropriate quantity to adjust the pH Preservative
Appropriate quantity 0.1-3 g/L Ionophore Effective quantity 0-200
mg//L Distilled water qsf Qsf 1 L
[0124] The present invention also proposes an apparatus for
implementing the method according to the invention which is
characterized by: [0125] an analysis tank which is able to receive
said analysis solution; [0126] a means for measuring the level of
hemoglobin present in said analysis solution by spectrophotometry
in said tank; [0127] a means for sampling said analysis solution;
[0128] a means for optical measurement on said sample in order to
produce a leucocyte analysis.
[0129] According to a second object, the present invention relates
to an optical device for an automatic apparatus for the automatic
analysis of a blood sample, particularly advantageously also for
the implementation of the method according to the first object of
the invention.
[0130] As mentioned above, certain sub-populations of leucocytes
can only be differentiated by optical measurements, for example a
measurement of the diffraction by the cell at one or more angles,
or a measurement of the absorbance of the cell. The optical systems
for characterization of a blood cell have a common base in which a
light source is located emitting a light beam, an optical tank in
which the blood cells cross the light beam, a system for adjustment
of the light beam to the flow of cells and means for measuring the
light originating from the optical tank after interception by the
cells. In particular in the case of leucocyte characterization, the
leucocytes move in a flow in the tank. They are illuminated therein
by a light beam focussed on the flow, which is called the sample
flow.
[0131] Such devices are costly: in particular, the lasers used as
light sources, which are also bulky and generally require a thermal
dissipation system; the laser diodes, like the lasers, require
costly alignment systems. The light beams emitted by these sources
have a transverse distribution of light which is approximately
Gaussian in shape. Thus, the intensity is only approximately
constant and maximal in a narrow and central part of the ray. The
alignment systems allow this central part to be aligned with the
sample flow. Moreover, the width of the sample flow must not exceed
that of this central part, and the closer these two widths are, the
greater the precision of the alignment system must be. As a result,
it is necessary to reduce the width of the sample flow as much as
possible.
[0132] The sample flow containing the blood cells to be counted
and/or to be differentiated must be narrower the more the light is
focussed. Thus, a flow is used in which the width of the section is
less than 50 .mu.m, which must cross the light beam which is itself
focussed into a narrow beam with a larger section than that of the
sample flow. This requires a particularly precise and therefore
costly system for injection of the flow into the optical tank. In
the prior art, such a result is obtained using a hydrofocus type
system (abbreviation of the English expression "hydrodynamic
focusing"). The sample flow is surrounded with a sleeving flow. An
injector for the sample flow is immersed in the centre of the
sleeving flow. The sample flow thus created is widened or focussed
as it travels from the injector to the zone illuminated by the
light beam, so that it has, at this point, a desired width of
approximately 5 to 50 .mu.m in diameter. A single or a double
sleeving is sometimes necessary in order to achieve this
objective.
[0133] Moreover, as mentioned previously, given the level of
precision required, an adjustment system is essential in order for
the flow of cells to be coincident with the light beam. Two
approaches are possible: the flow of cells or the light beam can be
moved. If it is chosen to move the flow of blood cells, all of the
optical tank unit must be moved. When this option is adopted, the
tank is mounted on a translation table which ensures a precise and
uniform movement along two axes due to its ball bearings. Such a
precision mechanical assembly is quite costly. It is also possible
to move the light beam in order to make it coincident with the flow
of blood cells. This is generally achieved using several adjustable
prisms. This solution, which combines optical elements with
precision mechanics also involves high costs.
[0134] Moreover, when it crosses the light beam, the blood cell
deflects the trajectory of the light rays. The intensity and the
angle of the deflected rays allow information on the cell type to
be obtained. Two ranges of angles are generally used: narrow angles
less than ten degrees with respect to the optical axis and wide
angles approximately perpendicular to the optical axis. In the
range of the narrow angles, two items of information are useful:
the losses in the axis and the diffraction. Perpendicular to the
optical axis, the diffusion and the fluorescence are generally
measured. For the two ranges of angles, the light must therefore be
distributed into two different channels. This is generally achieved
with dichroic mirrors or with interference filters. The optical
components are both produced by depositing thin films on a glass
substrate. They have good efficiency but a great disparity exists
between one filter and another and their lifetime is limited. They
must therefore be regularly replaced.
[0135] All these generally bulky devices are also fragile and
require maintenance, which is also very costly. Such devices are
therefore restricted to analytical laboratories which are large
enough to be able to invest in such automatic apparatuses.
[0136] The purpose of the invention is to propose a device for
leucocyte differentiation and/or leucocyte counting which is
simpler and more economical both to produce and to maintain,
allowing the use of automatic apparatuses, equipped with the
device, by smaller laboratories, while retaining adequate quality
of measurement.
[0137] According to the second object of the invention, an optical
device for counting and/or the differentiation of leucocytes in an
automatic blood analyzer is proposed, characterized in that it
comprises a light source of the electroluminescent diode type in
order to illuminate a blood sample circulating in the optical tank
according to an injection axis, using a source light beam. Such a
diode allows a light beam to be obtained which is more homogeneous
over the width of its section and therefore of a larger and more
homogeneous reading zone.
[0138] Preferably, the diode emits light the wave length of which
is less than 600 nanometers, and still more preferably less than
500 nanometers. Such a wavelength allows a better diffraction
efficiency, therefore better precision for measurements using
diffraction.
[0139] Moreover, the width of the beam emitted by the optical
device, i.e. the source beam which illuminates the sample flow, is
advantageously comprised between 50 and 200 microns (.mu.m), close
to the injection axis, which allows illumination of a wider sample
flow, while allowing adequate precision in the measurements carried
out. Yet more advantageously, this width is comprised between 90
and 120 microns. Such a flow width is in particular permitted by
the use of electroluminescent diodes.
[0140] Preferably, the source light beam is emitted approximately
in the direction of the tank, approximately transversely to the
direction of flow of the sample. A transparent slide designed so
that the source beam passes through it between two opposing
surfaces, which is rotatably mounted and arranged between the diode
and the tank can allow the light beam to be moved in a transverse
direction, thanks to its double refraction when it passes through
the slide. The rotation of the slide allows modification of the
angle of incidence of the beam on the slide, and thus adjustment of
the value of the transverse shift. Preferably, the transparent
slide is rotatably mounted about an axis which is approximately
parallel to the movement of the blood sample in the tank.
[0141] Beyond the optical tank, means for separation by Fresnel
losses are advantageously used for an incident resulting light beam
originating from the source light beam, thus separating said beam
into an axially-resulting beam and at least one beam resulting from
loss constituted by Fresnel losses whilst passing through the
separation means. The separation means comprise at least one
separation surface which is a surface in a transparent separation
material, the axial beam having passed through the transparent
material and the beam originating from the Fresnel losses having
been reflected by the separation surface, said surface being
slanted in relation to the light beam beyond the tank. A single
inexpensive glass: slide can serve as separation means. Moreover it
has a virtually unlimited and maintenance-free lifetime, unlike
dichroic mirrors or interference filters.
[0142] The device can also comprise an apparatus for measuring the
light of the axially-resulting beam and at least one other
apparatus for measuring the light of at least one beam originating
from the Fresnel losses. These measuring apparatuses can in
particular comprise means for measurement of either the
fluorescence, the light losses close to the axis or the diffraction
close to the axis. It can also comprise means for measuring the
diffraction of the light beam at wide angles by the sample in the
tank. By way of example, these wide angles can be angles comprised
between 60.degree. and 150.degree..
[0143] The device can also comprise, in the path of the beam in
front of the tank, at least one diaphragm blocking spurious
light.
[0144] The invention also relates to a haematology apparatus, in
particular an automatic blood analyzer equipped with such a
device.
[0145] According to a third object, the present invention also
relates to a flow-through optical tank for an optical device
suitable for the counting and differentiation of leucocytes, for
example a flow cytometer, as well as an analysis apparatus equipped
with such a tank. The aim of the invention is to propose a tank
which is simpler and more economical both to produce and to
maintain, allowing the use of automatic apparatuses equipped with
this tank by smaller laboratories, while retaining an adequate
quality of measurement.
[0146] According to the invention, a flow-through tank for an
optical device for the counting and differentiation of leucocytes
in an automatic blood analyzer, is characterized in that in an
analysis zone of the tank, the section of the tank has at least one
transverse dimension comprised between 1 and 5 millimetres. This
section can be approximately rectangular and the transverse
direction can be measured on one and/or the other of the sides of
the rectangle.
[0147] Such a tank can thus be produced, at least partially, from
an injected plastic material. Such a tank is produced in a
particularly advantageous manner compared to the tanks of the prior
art, generally formed of quartz walls assembled by bonding.
[0148] The tank can also comprise at least one lens moulded in one
piece with the tank. This at least one lens can comprise a lens
envisaged to be arranged laterally in relation to an optical axis.
It can comprise a hemispherical lens.
[0149] The tank can comprise along an optical axis, a window for
the introduction of a light beam and a window for the beam to exit.
At least one window can be moulded in one piece with the tank
and/or be an insert in a transparent material, for example quartz
or glass.
[0150] The tank can advantageously comprise an injector for a
sample flow and means for forming a sleeving flow around the
injection flow. The injector can comprise an outlet orifice the
diameter of which is comprised between 20 microns and 150 microns,
allowing a sample flow to be obtained which is noticeably larger
than the flows of the prior art. By contrast to the devices of the
prior art, it is not the sleeving flow which dictates the width of
the sample flow by stretching it, but the shape and the section of
the injector outlet. The sleeving flow therefore does not play an
active role, but merely a passive role, in particular, for example
for centering of the sample flow in a wide tank.
[0151] According to a first embodiment, this injector can be formed
in one piece in a more or less rigid material. This material can
be, for example, a stainless steel, a ceramic, synthetic ruby or a
plastic material or several of these materials.
[0152] According to a second embodiment, this injector can comprise
a rigid structural tube, for example made of metal, for example
made of stainless steel, and inside the structural tube, a plastic
sheathing tube ending in a nozzle formed in one piece with the
sheathing tube. The plastic material of the injector can be a
polytetrafluoroethylene, which allows the sample to circulate more
easily in the tube and reduces the risk of fouling up.
[0153] The invention also relates to an injector for a tank
according to the invention, which injector is produced according to
one of these embodiments.
[0154] The invention also relates to a haematology apparatus, in
particular an automatic blood analyzer, equipped with a tank
according to the invention.
[0155] According to a fourth object, the present invention also
relates to a hydraulic device for a haematological analysis
apparatus, which is simpler and more economical both to produce and
to maintain and which allows the use of automatic apparatuses,
equipped with such a device, by smaller laboratories, while
retaining an adequate quality of measurement. The present invention
also relates to an analysis method suited to such a device.
[0156] The present invention thus proposes a hydraulic device for a
blood analysis apparatus, in particular an automatic apparatus,
comprising means for injecting under pressure a sample flow into a
flow-through optical tank and for creating a liquid sleeving flow
around the sample flow, with a sleeving liquid, characterized in
that it comprises means for adjusting a flow rate of the sample
flow with respect to the flow rate of the sleeving liquid. Such
adjustment can make it possible to maintain homogeneous and
approximately non-turbulent flows in the tank.
[0157] The injection means can comprise syringes, a hydraulic
circuit and solenoid valves. These means can comprise means for
injecting the sample under pressure relative to the sleeving
flow.
[0158] This device can advantageously comprise means for forming a
piston for the sample injected with a displacement liquid. Such a
displacement liquid makes it possible to use only a small sample
sufficient for the analysis, the rest of the liquid required for
the injection being a liquid available in the analysis apparatus,
and not as precious as the sample.
[0159] The sleeving is particularly useful when using a tank with a
wide section while maintaining a small section for the sample flow.
As one of the means for adjusting the sample flow in relation to
the sleeving flow, the device can advantageously comprise means for
adjusting a flow rate of the displacement liquid with respect to
the flow rate of sleeving liquid. The adjustment means can comprise
means for a pressure drop in a branch circuit for the displacement
liquid and/or means for a pressure drop in a branch circuit for the
sleeving liquid. For example, the pressure drop means can be chosen
from a known length of a calibrated tube, a fixed hydraulic
resistance and a variable resistance.
[0160] The hydraulic device can comprise only one motorization, for
example a single electric motor, in order to generate the sample
flow and the sleeving flow simultaneously. Moreover, it can
comprise at least two syringes in order to generate the sample flow
and the sleeving flow, the syringe pistons being firmly attached to
each other. They thus have a common movement and the sample and
sleeving flows are indeed simultaneous.
[0161] In particular, a hydrofocus tank from the prior art can be
used with a circuit such as described previously according to the
invention, the injection of the sample into this tank can take
place without pressure relative to the sleeving flow.
[0162] According to the invention, a method for the analysis of a
blood sample in a flow-through cytometer is also proposed,
characterized in that a blood sample is injected, optionally under
pressure, into a flow-through tank of the cytometer, the sample
forming a sample flow there and a liquid sleeving flow is created
around the sample flow, with a sleeving liquid, characterized in
that the flow rate of the sample flow is adjusted with respect to
the flow rate of the sleeving liquid.
[0163] In particular, it is possible to introduce the sample into
an injection branch of a hydraulic circuit, and to introduce
upstream of the sample in the injection branch, a displacement
liquid, the displacement liquid serving to push the sample during
its injection into the tank. This displacement liquid can be chosen
from a reagent and a diluent, preferably a reagent. There is
therefore no point in providing a liquid other than that which is
strictly necessary for the preparation of the sample with a view to
its analysis or analyses.
[0164] It is also possible to create around the sample flow in the
tank, a sleeving flow with a sleeving liquid. This sleeving liquid
can also be chosen from a reagent and a diluent, preferably a
diluent. In this case also, there is not point in providing a
liquid other than those which are strictly necessary for the
preparation of the sample with a view to its analysis or
analyses.
[0165] In the case where a hydrofocus method, or a tank according
to the third object of the invention, is used, it is advantageous
to adjust the flow rate of displacement liquid with respect to the
flow rate of the sleeving liquid, for example by introducing a
pressure drop in a branch circuit for a displacement liquid and/or
pressure drop means in a branch circuit for a sleeving liquid.
[0166] In a method according to the invention, in particular for a
tank according to the third object of the invention, it can easily
be provided that the blood sample has a dilution rate of at least
1/100.sup.th. In fact, in such a method, the sample can be
introduced under pressure relative to the sleeving liquid, into the
tank, at a velocity greater than that of the methods of the prior
art, and with greater section widths for the sample flow in the
tank. Thus, without increasing the analysis time, for a
differentiation and a counting of the leucocytes, a dilution rate
can be used which is identical to that used conventionally for the
measurement of haemoglobin, in particular dilution rates comprised
between 1/100.sup.th and 1/500.sup.th, particularly between
1/160.sup.th and 1/180.sup.th.
[0167] The invention also relates to a haematology apparatus, in
particular an automatic blood analysis apparatus, characterized in
that it comprises a hydraulic device according to the
invention.
[0168] The present invention will be better understood and other
advantages will become apparent in light of the following
description of embodiments, which description is made in particular
with reference to the attached drawings in which:
[0169] FIG. 1 diagrammatically illustrates an example of equipment
according to the first object of the invention;
[0170] FIGS. 2a-2e are graphs of linearity tests of the measurement
of haemoglobin by spectrophotometry according to the method of the
invention;
[0171] FIGS. 2f-2i are corresponding cytographs;
[0172] FIG. 3 is a diagrammatic view of an automatic apparatus for
analysis of a blood sample using a hydraulic device according to
the fourth object of the present invention;
[0173] FIG. 4 is a diagrammatic longitudinal view of an optical
device unit according to the second object of the invention;
[0174] FIG. 5 is a more detailed diagrammatic longitudinal view of
the optical device of FIG. 4, in a plane perpendicular to that of
FIG. 4;
[0175] FIG. 6 is a perspective view of an optical tank according to
the third object of the invention;
[0176] FIG. 7 is a longitudinal section view of a first embodiment
of an injector for an optical tank according to the invention;
[0177] FIG. 8 is a longitudinal section view of a second embodiment
of an injector for an optical tank according to the invention;
[0178] FIG. 9 is a longitudinal section view of one end of the
injector of FIG. 8;
[0179] FIG. 10 is a longitudinal section view of a tank
illustrating a method of the prior art for injecting the blood
sample into the tank; and
[0180] FIGS. 11a-11c are graphs illustrating results obtained with
an automatic apparatus using the method of the invention and using
a cytograph with the optical device and tank according to the
invention.
[0181] FIG. 1 diagrammatically illustrates a single dilution and
analysis tank 1 which can be supplied with a blood sample 2 to be
analyzed, a diluent 3 and a reagent 4 together forming an analysis
solution. This tank 1 is equipped with means for measuring by
photometry 5 the haemoglobin level in said analysis solution and
means for measuring 6 the resistivity of said analysis solution in
order to count the total number of leucocytes. Means are generally
provided for taking a fraction of the analysis solution from the
analysis tank 1 and for injecting it into an optical tank 7
equipped with optical measurement means 8 (for example a flow
cytometer) for an analysis of the leucocytes. According to the
example chosen, means are also provided for taking a fraction of a
pre-solution constituted by the sample of blood and diluent, and
introducing it into a counting and dilution tank 9 equipped with
means for measuring the resistivity 10 of said fraction in order to
count the erythrocytes and platelets. The equipment is
conventionally equipped with heating means in order to obtain a
thermostatically-controlled temperature of approximately 35.degree.
C. This temperature allows optimal lysis reaction time and quality
of the erythrocytes.
[0182] The equipment operates in the following manner:
[0183] one aliquot of blood (15.6 .mu.l) is injected into the
analysis tank 1 and diluted with 2 ml of diluent so as to form an
analysis pre-solution; the dilution rate is 1/130.sup.th;
[0184] a very small fraction (approximately 20 .mu.l) is taken from
this analysis pre-solution and deposited in the tank 9 for counting
the erythrocytes and platelets;
[0185] 0.7 ml of reagent is then added to the remaining
pre-solution in the analysis tank 1: the lysis lasts for
approximately 10 seconds (in order to destroy the erythrocytes,
form and stabilize the oxyhemoglobin complex), the analysis
solution thus formed has a final dilution rate of approximately
1/173.sup.rd; a fraction of said analysis solution is taken and
injected into the optical tank 7 where the analysis of the
leucocytes can take place (counting and/or differentiation of the
leucocytes by sub-populations); simultaneously in the analysis tank
1, the leucocytes are counted by a resistivity measurement and the
haemoglobin by a measurement by absorbance at the wave length of
the oxyhemoglobin complex formed.
[0186] An optical device according to the invention, particularly
suitable for a leucocytic analysis of an analysis solution having a
dilution rate lower than 1/100.sup.th is described below, more
particularly suitable for a dilution comprised between 1/160.sup.th
and 1/180.sup.th Conventionally, a dilution rate of 1/160.sup.th is
considered to be lower than a rate of 1/100.sup.th.
[0187] Of course variant embodiments of the method and of the
equipment described above are possible:
[0188] for the equipment: means can be provided for separately
introducing the lysis compound, the leucoprotective compound and
the compound stabilizing the complex formed with the haemoglobin in
the analysis tank 1, and therefore rather more in the form of a
mono-reagent; the means 6 for measuring the resistivity of the
analysis solution are optional; the total number of leucocytes
being able to be obtained by optical analysis of the analysis
solution; similarly the counting tank 9 and the means for measuring
10 the resistivity in this tank can be provided only if a complete
analysis of the blood sample is desired;
[0189] likewise for the method: the introduction of the reaction
compounds can be envisaged independently or collectively in place
of a mono-reagent, the introduction being able to be carried out
simultaneously or successively; the previous stage of counting the
erythrocytes and platelets and the stage of global counting of the
leucocytes can be omitted; moreover, two successive dilutions of
the blood sample can be carried out: a first dilution which is
particularly suitable for a leucocyte differentiation
(approximately to 1/80.sup.th) as takes place in the known standard
hydrofocus-type cytometer, from which the fraction required for
this leucocyte differentiation is taken, then at a second moment in
time a second dilution suitable for measurement of the haemoglobin
(comprised between 1/100.sup.th and 1/500.sup.th) as is possible
with the known spectrometers.
[0190] According to yet another variant, the tank 1 can serve at a
second moment in time to carrying out counting the erythrocytes and
platelets after cleaning, by filling the tank with a sample waiting
in a syringe needle.
[0191] The results obtained will now be described with a specific
example of (mono)-reagent according to the invention:
[0192] A mono-reagent is prepared using the Eosinofix.RTM.
formulation from the company ABX marketed for leucocyte
determination in flow cytometry and containing for this purpose a
compound for lyzing the erythrocytes and a leucoprotective compound
(cf. patent EP0430750 by ABX). According to the invention, a
compound stabilizing the haemoglobin complex was added.
Measurement of the Haemoglobin by Spectrophotometry:
[0193] Linearity tests were carried out using a spectrophotometer
at 542 nm. The graphs are shown in FIGS. 2a-2e. They represent the
haemoglobin concentrations measured in relation to the expected
concentrations. More specifically: [0194] FIG. 2a corresponds to a
reference lysis for measurement of the haemoglobin by
spectrophotometry (LMG.RTM. sold by the company ORPHEE); [0195]
FIGS. 2b, 2c and 2d correspond to the mono-reagent according to
embodiment No. 4 with, as stabilization agent of the haemoglobin
complex, respectively Tiron, DDAPS and imidazole; and [0196] FIG.
2e corresponds to the method of the invention implemented using as
mono-reagent Eosinofix.RTM. alone, i.e. [0197] containing no
stabilization agent of the haemoglobin complex according to the
present invention.
[0198] For the three tests carried out according to the invention,
a positive linearity test is obtained for each with a correlation
coefficient R.sup.2 of 1.+-.10.sup.4 (shown in the figure). This
result is in accordance with that obtained with the reference lysis
of FIG. 2a. This means that the method of the invention does indeed
allow measurement of a real haemoglobin level in a blood
sample.
[0199] By contrast, as is seen in FIG. 2e, with the reagent
(Eosinofix) without a hemoglobin stabilizer, a linear relationship
is not obtained. This means that this reagent alone cannot be used
to measure a haemoglobin level.
Leucocyte Differentiation by Flow Cytometry
[0200] FIGS. 2f to 2i are cytographs obtained using a BD
FACScan.RTM. flow cytometer, corresponding respectively to
Eosinofix alone and Eosinofix to which DDAPS, Tiron and imidazole
are added. In these figures, it is seen that the differentiation of
the sub-populations is indeed achieved and in a manner which is
comparable to a standard reagent for leucocyte differentiation
(matrix obtained with Eosinofix in FIG. 2f).
[0201] Reference can also be made to the cytograph of FIG. 11b
(described below) in particular obtained with a cytometer according
to the invention.
[0202] The hydraulic device according to the fourth object of the
invention will now be described.
[0203] FIG. 3 partially represents the diagram of a hydraulic
system 100 and some of the equipment of an automatic blood analyzer
20, in so far as it allows an understanding of the hydraulic device
according to the invention.
[0204] The automatic apparatus illustrated in FIG. 3 in particular
comprises a needle 101 for sampling blood to be analyzed in a tube
which was used for its storage and its transport to the automatic
apparatus. The blood taken is poured by the needle in the form of a
sample into a tank 102. The tank 102 is in particular designed for
the dilution and/or the lysis of the erythrocytes of the blood
sample. All or part of the sample, before or after dilution, can be
taken with a view to analysis in another part of the automatic
apparatus, for example in a device 120, described below. A device
for analysis of the haemoglobin 110 (a spectrophotometer for
example) is arranged close to the tank 102. A store 103 for a
dilution product and a store 104 for a reagent, in particular a
lysis reagent are connected to the tank 102 via the hydraulic
circuit 100.
[0205] Another analysis device 120 is more specifically dedicated
to the counting and differentiation of the leucocytes, for example
on the whole or part of the sample taken from the tank 102.
Hereafter sample will also refer to this whole or this part. The
device for analysis of the leucocytes 120 in particular comprises
an optical device 200 and an optical tank 300. The optical tank is
connected to the tank 102 via the hydraulic circuit.
[0206] A set of syringes allows the movement of the liquids in the
hydraulic circuit. Of these syringes, a syringe 105 dedicated to
the diluent and a syringe 106 dedicated to the reagent are
represented so that the invention is well understood. Other
syringes which are not represented because they are not necessary
in order to understand the invention can complete the device.
[0207] Besides the pipes for the circulation of the liquids, the
hydraulic circuit comprises solenoid valves for the change-over of
different circuits in the hydraulic circuit 100, according to its
use at a given moment of the analysis. Eight solenoid valves
111-119 of the solenoid valves of the hydraulic circuit 100 are
illustrated in FIG. 3. Each solenoid valve comprises two positions,
each labelled respectively with the letter A or B.
[0208] The design of the hydraulic circuit as will be described
below, allows the use of only one motorization M for the syringes
illustrated. The same motorization can also be used for other
syringes. Thus, the pistons of the syringes 105, 106 are firmly
attached to each other. Their movement is therefore simultaneous,
either pushing P, when they are driven into the respective cylinder
of each syringe, or pulling T when they are withdrawn.
[0209] The arrangement and then the hydraulic operation of the
automatic apparatus will now be described.
[0210] The tank 300 comprises an external body 301 and an injector
302, inside the body 301, a sleeving volume 303 is formed between
the body and the injector.
[0211] The hydraulic circuit 100 comprises: [0212] an injection
branch 131 which extends upstream of the injector, between the
injector and the valve 111; [0213] a sample branch 132 which is
connected at a sample branching point 142 to the injection branch
and extends to the tank 102; [0214] a suction branch 133 which is
connected at a suction branching point 143 to the injection branch,
upstream of the sample branching point 142, via the valve 113 and
extends to a vacuum source 107, for example a syringe or a
peristaltic pump; [0215] a discharge branch 134 which is connected
at a discharge branching point 144 to the injection branch,
upstream of the suction branching point 143, and extends to the
reagent product store 104; [0216] a sleeving branch 135 which
extends upstream of the body 301 and connects the sleeving volume
and the valve 115; [0217] a dilution branch 136 which extends
between the valve 116 and a use 108 for the diluent via the valve
115; [0218] a diluent branch 137 which extends between the diluent
store 103 and the valve 116; [0219] a reagent branch 140 which
extends between the reagent store 104 and the valve 117; [0220] a
reaction branch 141 which extends between the valve 117 and a use
109 for the reagent via the valve 111; [0221] a draining branch 138
for the tank 102 which extends between the tank 102 and the vacuum
source 107 via the valve 118, the sample branch 132 being connected
with the draining branch between the tank 102 and the valve 118,
and the suction branch being connected to the outlet branch 132
beyond the valve 118 in relation to the tank; and, [0222] an outlet
branch 139 which connects the downstream of the tank 300, via the
valve 119, to a waste tank, for example at atmospheric pressure or
via a suction source, a syringe or a peristaltic pump.
[0223] In a first position 116A of the valve 116, the dilution
syringe 105 is in communication with the diluent store, so that a
pulling movement T allows the syringe 105 to be filled with
diluent.
[0224] In a first case the dilution syringe containing diluent,
with the valve 116 being in its second position 116B which connects
the syringe 105 to the dilution branch 136 and the valve 115 being
in its first position 115A which connects the dilution branch to
the use 108 for the diluent, a pushing movement P allows the
diluent to be moved to this use 108, for example in the tank 102,
for example for a dilution of the whole sample.
[0225] In a second case, with the valve 116 being in its second
position 116B and the valve 115 being in its second position 115B
which connects the dilution branch to the sleeving branch 135, a
pushing movement P allows the diluent to be moved into the optical
tank 300, in order to form a sleeving flow there. The usefulness of
this sleeving flow in the context of the invention will be analyzed
in a description of the tank 300 below.
[0226] The valve 117 being in a first position 117A which connects
the syringe of reagent to the reagent store 104, and the valve 114
being in a first position 114A which shuts off the discharge branch
134, a pulling movement T allows the reagent syringe 106 to be
filled with reagent.
[0227] In a first case, the reagent syringe containing reagent,
with the valve 117 being in its second position 117B which connects
the reagent syringe 104 to the reaction branch 141 and the valve
111 being in a first position 111A which connects the reaction
branch to the use 109 for the reagent, a pushing movement P allows
the reagent to be moved to this use 109, for example in the tank
102, for example for a lysis of the whole sample.
[0228] In a second case, the valve 117 being in its second position
117B and the valve 111 being in its second position 111B which
connects the reaction branch 141 to the injection branch 131, the
reagent syringe 106 is directly connected to the injector 302.
[0229] The valve 118 being in a first position 118A which isolates
the suction branch 133 from the sample branch 132 through the
draining branch, the valve 112 being in a first position 112A which
connects the upstream part to the downstream part of the sample
branch 132, the valve 113 being in a first position 113A which
connects the downstream part to the upstream part of the suction
branch 133, therefore to the vacuum source 107, the sample to be
analyzed is sucked into the injection branch 131, between the
sample branching point 142 and the suction branching point 143.
[0230] The discharge branch 134 comprises a variable or calibrated
fluid resistance 150.
[0231] When the diluent syringe 105 contains diluent, the reagent
syringe 106 contains reagent and a blood sample to be analyzed is
in the injection branch 131; and when the valves 112, 113 are in
their second positions 112B, 113B which isolate the upstream part
and the downstream part from their respective arms; and when the
valves 115, 116 are in their second positions 115B, 116B which
connect the diluent syringe 105 to the sleeving volume 303; when
finally the valves 111, 117 are in their second positions 111B,
117B which connect the reagent syringe 106 to the injector 302 and
the valve 114 is in its second position 114B; a single pushing
movement P generated by the single motorization M, allows the
driving of the diluent, the reagent and the blood sample in the
direction of and through the tank 300, while a part of the reagent,
which is a function of the fluid resistance 150, is returned to the
reagent store 104.
[0232] The resistance 150 in particular allows adjust of the flow
rates of the sleeving and displacement liquids one with respect of
the other. This allows these flow rates to be adapted to the
different functions of these liquids. In particular, this allows
similar flow velocities to be obtained for the sleeving and the
sample in the analysis zone 304 when a standard hydrofocus tank is
used.
[0233] In particular, the discharge branch 134 and the arrangements
described previously make it possible to use a single motorization
and therefore to reduce in particular the cost of an automatic
analysis apparatus, as well as its bulk.
[0234] The diluent forms, in an analysis zone 304 of the tank 300 a
sleeving flow for the sample (see in particular FIGS. 4 and 5). The
reagent, situated upstream of the sample in the injection branch
131, serves as a displacement liquid, i.e. it allows the piston
movement of the reagent syringe to be transmitted to the sample.
Thus, there is no point in filling the reagent syringe with the
sample in order to be able to undertake its analysis. Thus, even a
sample of small volume can be analyzed, and all of this sample can
be injected and analyzed without some of it remaining in the
injection branch 131 or in the syringe 106.
[0235] Of course, other syringes, valves and branches, not
represented in FIG. 3, can make up the hydraulic circuit 100, in
order for the automatic analysis apparatus 20 to operate fully and
well.
[0236] The optical device 200 according to the invention will now
be described, in particular with regard to FIGS. 4 and 5.
[0237] The optical device comprises an approximately monochromatic
light source 201. This light source is an electroluminescent diode.
The light is principally emitted along an optical axis X200. The
optical axis X200 is arranged approximately perpendicular to an
injection axis X300 for movement of the sample in the optical tank
300. The two axes X200 and X300 together define an optical
plane.
[0238] In order to prevent the source light beam 311 produced by
the source 201 from being polluted by spurious light, a set of
three diaphragms, is arranged, each one perpendicular, on the path
of the beam. The diaphragms 202 are pierced with holes the diameter
of which is approximately equal to the beam and is progressively
increased in each diaphragm in order to adapt it to the diameter of
the measurement beam as this diameter increases the further away
from the source 201 it is. The beam then passes through a focusing
device 203 constituted by one or more lenses.
[0239] Beyond the focusing device the beam encounters an adjustment
device which allows the optical axis to be moved in a plane
perpendicular to the injection axis X300, i.e. in a transverse
direction in relation to the movement of the sample in the tank. A
lateral shift of the beam can lead to a partial or no illumination
of the sample which has a direct influence on the analysis
result.
[0240] In the context of the example described, the adjustment
device is constituted by a transparent slide 220 rotatably mounted
about an axis X220. The axis 221 is approximately parallel to the
injection axis X300. If the slide is arranged perpendicular to the
optical axis X200, the beam passes through it without being
deflected. By contrast, if the slide forms an angle with the
optical axis, a double refraction, at entry and exit of the slide,
shifts the beam in a plane perpendicular to the adjustment axis
X220. The adjustment axis X220 being approximately parallel to the
injection axis X300, only a transverse shift is generated by the
refraction in the slide. The greater the thickness and/or the
refractive index of the slide and the more the slide is inclined
with respect to the optical axis the greater is the shift. Thus,
for a slide with a chosen thickness and refractive index, it is
sufficient to rotate the slide 220 about its axis X220 in order to
adjust the position of the beam relative to the sample which moves
in the analysis zone 304 of the optical tank 300. Such an
adjustment device is particularly economical compared to the
devices of the prior art, especially as a precise rotation is
generally easier to carry out than a precise translation, using
high-precision mechanics.
[0241] After having penetrated the tank and passed through the
sample, the source beam 211 at least partially becomes an
axially-resulting beam 212, which exits the tank approximately
along the optical axis. The axially-resulting beam 212 carries
information about the sample which it has passed through.
[0242] In order to allow simultaneous measurements of several of
these items of information it must be possible to analyze the beam
with several measurement apparatuses 222, 223. In particular, the
optical analysis relies on the detection of the light diffracted
according to two ranges of angles: narrow angles and wide angles.
In each of the ranges of angles, two different items of information
are used. It is therefore necessary to distribute the light in two
different channels for each range. Therefore means 205 for
separating the resulting beam 212 into two resulting beams 213, 214
are used. The separation means are mainly constituted by a beam
splitter 205. This beam splitter is a transparent glass slide. It
is arranged at 45 degrees to the optical axis. A secondary
axially-resultant beam 213, formed by the light which has passed
through the beam splitter, and a beam resulting from loss 214
formed by the Fresnel losses, i.e. by the light reflected by the
beam splitter, are thus produced.
[0243] Such a beam splitter has a very low cost compared to the
separation means used in the prior art in optical analysis devices
of this type. In particular, because it does not comprise any
additional reflective coating, it is virtually age resistant and
requires practically no maintenance. Given the multiple reflections
inside the slide and the polarization of the incident radiation of
the axially-resulting beam, between 5 and 15% of the energy is
reflected, the rest being transmitted in the form of the secondary
axially-resulting beam.
[0244] Between the tank and the beam splitter, the
axially-resulting beam 212 is rendered parallel by suitable means
206. Beyond the beam splitter, the resulting beams 213, 214 are
again focussed by respective suitable means 207, 208, with a view
to their analysis by the respective measurement apparatuses 222,
223.
[0245] In the example described, the measurement apparatus 222,
which analyzes the secondary axially-resulting beam 213 is an
apparatus for measurement of the diffraction close to the optical
axis by the blood cells (called an FSC measurement). In the example
described, the measurement apparatus 223, which analyzes the beam
produced by the Fresnel losses 214 is an apparatus for measurement
of the light losses in the axis (called an ALL measurement), i.e.
the obscuring of the light by the cells in the sample.
[0246] FIG. 5 diagrammatically represents a section of the tank in
a plane perpendicular to the injection axis X300 and containing the
optical axis X200. As is particularly illustrated in this figure,
the light reemitted laterally by the sample in a
laterally-resulting flow 315, focussed beyond the tank in a
measurement apparatus 224, is also analyzed.
[0247] An optical tank according to the invention, in particular
envisaged for use with a hydraulic circuit such as described
previously, will now be described, in particular with reference to
FIG. 6. The operation of this tank can be compared with a
hydrofocus-type operation of the prior art, which is represented
very diagrammatically in FIG. 10.
[0248] The tank 350 of FIG. 10 comprises a body 351, an injector
302 and an analysis zone 354. An internal transverse dimension D354
of the tank is approximately 250 microns. This dimension can be a
diameter, if the tank has a circular section, or one side, if it
has a square or rectangular section. As illustrated by the dotted
lines, a sleeving flow 362 is used to reduce in particular the
diameter of a sample flow 361, so that in the analysis zone 354 the
sample flow has, in the prior art, a diameter D361 of less than 50
microns.
[0249] The tank 300 according to the invention, illustrated in
FIGS. 4-6, comprises the body 301 and the injector 302, arranged
approximately coaxially along an injection axis X300. The analysis
zone 304 is arranged downstream of the injector.
[0250] The body is produced from an injected material, preferably
from a plastic material. Such a production method allows complex
shapes to be obtained. In particular, a lens 305 is moulded in the
body. This lens allows the light which is obscured, diffracted or
diffused by the blood cells to be collected.
[0251] This lens must have dimensions, in particular sufficient
diameter for the possible local inhomogeneities in the injected
material to be negligible in relation to these dimensions. In the
example illustrated, the lens 305 has a diameter of about 3 mm.
This injected lens is a lateral lens 305 which the
laterally-resulting beam 315 passes through. Moreover, the lateral
lens must allow the light to be collected in as many directions as
possible, i.e. with a directional field which is as large as
possible. Thus, the closer the lens is to the sample, the greater
the directional field. In the example illustrated, the lens is a
hemispherical lens, called a 90.degree. lens. Moreover, the lens
being a part of the wall of the tank, there is direct contact with
the liquid in the tank, i.e. there is no air space, with a low
refractive index, between the sample and the lens. This improves
the measurement.
[0252] In order to overcome the homogeneity deficiencies, glass is
used where the light is particularly focussed, for example a glass
of the BK7 type. This is the case in particular for the axial
windows 306, where the source beam 211 penetrates the tank and
where the axially-resulting beam 212 exits it.
[0253] In order to be able to produce an injected lens with such
dimensions, it is necessary, in the analysis zone, for the tank 300
to have at least comparable dimensions. Moreover these large
dimensions allow glass windows to be integrated into plastic walls,
while the tanks of the prior art, having small dimensions, are made
with walls entirely of glass or quartz. In the example illustrated
in particular in FIGS. 5 and 6, the lower section of the tank is
4.5 mm along the optical axis by 3 mm in the perpendicular
direction. This rectangular section with large dimensions
associated with a small volume of the sample, which transports the
blood cells to be analyzed, requires the use of a hydrodynamic
sleeving of the sample. By way of comparison, a tank of the prior
art has an internal transverse dimension D354 of the analysis zone
close to 250 microns.
[0254] Upstream of the analysis zone 304, the body 301 of the tank
surrounds the injector 302 and forms around the injector the
sleeving volume 303. The walls of the injector separate a flow 311
formed by the sample, inside the injector, from a sleeving flow
312, in the sleeving volume. The sample flow originates from the
injection branch 131 of the hydraulic circuit 100. The sleeving
flow originates from the sleeving branch 135 of the hydraulic
circuit. In the analysis zone, the two flows are in contact, remain
concentric and flow simultaneously in the tank.
[0255] In order to reduce the production costs of the automatic
apparatus, it can be advantageous to reduce the precision of the
production of the parts. As mentioned above, such an aim can be
achieved by creating a sample flow with a larger section.
[0256] However, if a technique of the prior art is used where the
sample flow is stretched by a sleeving flow, a sample flow with a
large section will be turbulent, which in particular adversely
effects the precision of measurements. Moreover the section of the
sample flow will be progressively reduced, which is the opposite of
the effect desired, which is to have a sample flow with a large
section. Such an aim is achieved by using the hydraulic circuit 100
according to the invention, described previously with reference to
FIG. 1. Such a circuit makes it possible to obtain independently
chosen velocities for the flow of the sleeving flow and for that of
the sample flow, in order that little turbulence appears in the
sample flow and that this turbulence has no notable effect on the
analysis result. The two flows can each be approximately uniform,
optionally laminar in certain appropriate velocity ranges.
[0257] Moreover, an injector 302 as illustrated in FIG. 7 or FIG. 8
also allows limitation of the turbulence in the sample flow.
Moreover, it allows a high velocity of injection of the sample into
the optical tank, while retaining its flow approximately
uniform.
[0258] An injector 302 as illustrated in FIG. 7 comprises a
structural tube 320, for example made of stainless steel ensuring
the stiffness of the injector. The structural tube is sheathed on
the inside with a tube 321 made of a plastic, for example a
polytetrafluoroethylene (PTFE). In the example illustrated the
structural and sheathing tubes are cylindrical. The sheathing tube
is extended, downstream of the structural tube, by a nozzle made of
the same plastic material. The fact of differentiating the
structural function of the structural tube and the injection
function of the nozzle, associated with the use of a plastic
material, allows sufficiently precise shapes to be obtained at a
low cost.
[0259] The nozzle has a section which is progressively narrowed
from an internal diameter D321 of the sheathing tube to an internal
diameter D323 of an outlet orifice 323 at a downstream end 324 of
the nozzle 322. In the example described, the downstream end 324 is
a cylinder with a length L324. The wall of the nozzle is initially
inwardly concave, then inflected to become inwardly convex, the
section of the nozzle thus being progressively narrowed from the
upstream to the downstream, from the diameter D321 to the diameter
D324. The concave surface is tangent to the inner surface of the
cylindrical sheathing tube. The convex surface is tangent to the
inner surface of the cylindrical end 324. In the example described,
the diameter D323 of the orifice 323 is approximately 60 microns,
the internal diameter D321 of the sheathing tube is approximately 1
millimetre, the length L322 of the nozzle is approximately 2.5
millimetres, that L320 of the structural tube is approximately 6
millimetres and that of the cylindrical end L324 approximately 200
microns.
[0260] An injector 302 such as that illustrated in FIGS. 8 and 9 is
in a single piece and made of a single substantially stiff
material. This material can be, for example, stainless steel, a
ceramic, a synthetic ruby or a plastic material. The plastic
material can advantageously be a polytetrafluoroethylene. The
injector comprises an approximately cylindrical tube 331 which is
extended downstream by a nozzle 332.
[0261] The nozzle progressively narrows inwardly, from an internal
diameter D331 for the tube 331, to an internal diameter D333 of an
outlet orifice 333 for the sample, at a downstream end 334 of the
nozzle 332. In the example illustrated, the narrowing takes place
according to a truncated cone open at an angle preferably comprised
between 9 and 10 degrees. Beyond the truncated cone and up to the
outlet orifice 333, the diameter remains constant in a cylindrical
part 335, with a length L335 and a diameter D333.
[0262] On the exterior of the nozzle, its external diameter is
progressively larger according to a truncated cone open at an angle
comprised between approximately 8 and 9 degrees, then, in the
noticeably more reduced end according to a truncated cone open at
an angle A334 comprised between approximately 35 and 45 degrees, to
an external diameter D334 around the outlet orifice 333. D334 is
approximately 3 to 4 times larger than D333.
[0263] By way of example D333=60 .mu.m, D334=200 .mu.m and
A334=40.degree..
[0264] Thanks to the different arrangements described previously,
it is possible to obtain a high injection velocity. Thus, in the
example described it is possible to inject a sample of more than
200 microlitres in less than 10 seconds. In particular, such an
injection rate makes it possible to use a high rate of dilution of
the blood sample, without increasing the duration of the analysis
compared to automatic apparatuses of the prior art. In particular,
the same dilution, for example 1/160.sup.th, can be used for the
analysis of the haemoglobin by the device 110 (see FIG. 3) and for
the analysis of the leucocytes by the optical device 120, instead
of 1/80.sup.th generally used for the analysis of the
leucocytes.
[0265] FIGS. 11a-c illustrate the results obtained using the method
and the equipment according to the first object of the invention,
said equipment using an optical tank 7 according to the third
object of the invention and an optical device 8 according to the
second object of the invention. FIG. 11a shows a positive linearity
test of the haemoglobin measurement and therefore demonstrates the
possible and reliable measurement of the haemoglobin level of a
blood sample according to the invention. FIG. 11b shows an optical
matrix obtained from a test sample of blood with 30% eosinophils to
which the formulation according to the invention has been added. On
this matrix, the five sub-populations are present and
differentiated (groups delimited in the cytograph: E for
eosinophils, N for neutrophils, M for monocytes, B for basophils
and L for lymphocytes). FIG. 11c shows the positive linearity test
of the measurement by resistivity of the level of leucocytes.
[0266] These figures show that thanks to the invention it is
possible to carry out an analysis of at least the level of
haemoglobin and the level of leucocytes and a leucocyte
differentiation using the formulation according to the invention,
in particular in the form of a mono-reagent.
[0267] Of course, the invention is not limited to the examples
which have just been described and numerous modifications can be
applied to these examples without exceeding the scope of the
invention.
[0268] For example, products other than the diluent or the reagent
can be used in order to form respectively the sleeving flow and the
fluid piston, particularly if they are available in the automatic
apparatus for other uses.
[0269] In addition, instead of being arranged only on the injection
circuit, a fluid resistance can be arranged on the sleeving circuit
or on both of these simultaneously. This can occur as a function of
the given maximum flow rate via the means for displacement of the
liquids intended respectively for displacement or sleeving.
[0270] Several or all of the lenses of the optical tank and/or of
the optical device can thus be produced by injection with the body
of the tank, instead of a single one as illustrated previously. In
particular, the glass windows can be injected. Particularly if the
inhomogeneities in the injected material are more or less
negligible with regard to the precision desired for the
measurements.
[0271] An adjustment device and/or the separation means described
previously can be used independently of each other and optionally
with a light source other than an electroluminescent diode.
* * * * *