U.S. patent application number 10/081118 was filed with the patent office on 2002-12-05 for reagent and process for the identification and counting of biological cells.
Invention is credited to Champseix, Henri, Lefevre, Didier, Veriac, Sylvie.
Application Number | 20020182623 10/081118 |
Document ID | / |
Family ID | 8860367 |
Filed Date | 2002-12-05 |
United States Patent
Application |
20020182623 |
Kind Code |
A1 |
Lefevre, Didier ; et
al. |
December 5, 2002 |
Reagent and process for the identification and counting of
biological cells
Abstract
The invention relates to a reagent and a process for the
identification and counting of biological cells in a sample. This
reagent comprises a cell lysing agent selected from at least one
detergent in a concentration capable of specifically lysing a given
type of cells in the sample, and a stain capable of marking the
intracellular nucleic acids of the remaining unlysed cells.
Application in particular for the identification and counting of
cells using an automated analysis system based on flow
cytometry.
Inventors: |
Lefevre, Didier; (Saint
Clement De Riviere, FR) ; Veriac, Sylvie;
(Montpellier, FR) ; Champseix, Henri; (Saint Gely
Du Fesc, FR) |
Correspondence
Address: |
George R. Pettit
Connolly Bove Lodge & Hutz LLP
Suite 800
1990 M Street, N.W.
Washington
DC
20036-3425
US
|
Family ID: |
8860367 |
Appl. No.: |
10/081118 |
Filed: |
February 25, 2002 |
Current U.S.
Class: |
435/6.16 ;
435/40.5; 435/7.21 |
Current CPC
Class: |
Y10T 436/2525 20150115;
Y10T 436/25125 20150115; Y10T 436/101666 20150115; Y10T 436/108331
20150115; G01N 33/5094 20130101; Y10T 436/107497 20150115; Y10T
436/10 20150115 |
Class at
Publication: |
435/6 ; 435/7.21;
435/40.5 |
International
Class: |
C12Q 001/68; G01N
033/567; G01N 033/48 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 23, 2001 |
FR |
102489 |
Claims
1. Reagent for the identification and counting of biological cells
in a sample, in particular in a blood sample, characterised in that
it comprises: a cell lysing agent selected from at least one
detergent in a concentration sufficient to lye specifically a given
type of cell in the sample, and a stain designed to mark the
intracellular nucleic acids of the unlysed remaining cell.
2. Reagent according to claim 1, characterised in that the cell
lysing agent comprising at least one ionic and/or non-ionic
detergent in a concentration capable of lysing erythrocytes.
3. Reagent according to claim 1, characterised in that the
detergent is selected from: primary amines, amine acetates and
hydrochlorides, quaternary ammonium salts, and trimethylethyl
ammonium bromide; amides of substituted diamines,
diethanolaminopropylamine or diethylaminopropylamide, amides of
cyclised diethylenetriamine; alkylaryl sulfonates, petroleum
sulfonates, sulfonated glycerides; cholamides, sulfobetaines; alkyl
glycosides, saponins; polyoxyethylene ethers and sorbitans,
polyglycol ethers.
4. Reagent according to claim 1, characterised in that the stain is
a fluorescent type stain.
5. Reagent according to claim 1, characterised in that the stain is
capable of combining specifically with the intracellular
ribonucleic acid and enhancing its fluorescence once it has
combined with the latter.
6. Reagent according to claim 1, characterised in that the stain is
selected from: thiazole orange or
1-methyl-4-[(3-methyl-2-(3H)-benzothiaz-
olylidene)methyl]quinolinium p-tosylate, thiazole blue,
4-[(3-methyl-2-(3H)-benzothiazolyl-idene)methyl]-1-[3-(trimethylammonium)-
propyl]quinolinium diiodide, 3,3'-dimethyloxacarbocyanine iodide or
3-methyl-2-[3-(3-methyl-2(3H)-benzothiazolylidene-1-propenyl]benzoxazoliu-
m iodide, thioflavine T, thioflavine T, the stains SYTO.RTM. and
TOTO.RTM. (TM Molecular Probes), ethidium bromide, propidium
iodide, acridine orange, coriphosphine O, auramine O, the stains
HOECHST 33258 and HOECHST 33342, 4',6-diamino-2-phenylindole
dihydrochloride (DAPI), 4',6-(diimidazolin-2-yl)-2-phenylindole
dihydrochloride (DIPI), 7-aminoactinomycin D, actinomycin D, and
LDS 751.
7. Reagent according to claim 1, characterised in that it also
comprises at least one membrane penetration agent capable of
promoting the penetration of the stain into the cells to be
marked.
8. Reagent according to claim 7, characterised in that the agent
promoting membrane penetration is an ionophore compound of the
protonophore and/or antibiotic type.
9. Reagent according to claim 1, characterised in that it also
comprises at least one membrane fixing agent present in a
concentration of 0.1% to 10% (w/v).
10. Reagent according to claim 9, characterised in that the
membrane fixing agent comprises at least one alcohol and/or an
aldehyde selected from paraformaldehyde and glutaraldehyde.
11. Reagent according to claim 1, characterised in that it also
comprises at least one compound selected from a complexing agent,
an inorganic salt and a buffer system.
12. Process for the identification and counting of biological cells
in a sample, in particular in a blood sample, characterised in that
it comprises the following operations: mixing and incubating the
sample with a reagent according to one of claims 1 to 11 in order
to effect, in a single stage, the lysis of cells of a given type,
in particular erythrocytes, the staining of the intracellular
nucleic acids, and the fixing of the nucleate cells; measuring the
resultant solution by flow cytometry using at least two measuring
parameters selected from resistive volume, axial luminous
diffraction, axial luminous transmission, orthogonal luminous
transfusion, and fluorescence; and classifying and counting the
nucleate cells in populations by means of the measured
parameters.
13. Process according to claim 12, characterised in that the
resistivity measurement is carried out by means of at least one
current selected from a continuous current and a pulsed or
alternating current.
14. Process according to claim 12, characterised in that the axial
luminous diffraction parameter is at least one parameter selected
from small angle diffraction and large angle diffraction.
15. Process according to claim 12, characterised in that the
classified nucleate cells are either mature or immature, normal or
abnormal cells.
16. Process according to claim 12, characterised in that the
classification of the nucleate cells is carried out by means of a
multidimensional analysis software program, with or without the use
of a software or other neuronal technique.
17. Process according to claim 12, characterised in that the sample
is a sample of human or animal blood.
18. Process according to claim 12, characterised in that the sample
is a sample of biological fluid or a suspension of cells of human
or animal origin.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to biological analyses and in
particular to blood analyses.
[0002] The invention relates more particularly to a reagent and a
process for the identification and counting of biological cells in
a sample, in particular in a blood sample.
[0003] The biological sample may be human or animal blood, or also
any other biological liquid or biological preparation.
BACKGROUND OF THE INVENTION
[0004] In the field of biological analyses the importance of the
determination and precise counting of different cell populations in
making a diagnosis has been recognised for a long time. In fact,
the appearance of abnormal equilibrium ratios among normal cell
populations in blood may be correlated with the appearance of
certain medical conditions, for example immune reactions,
inflammatory reactions, etc. Similarly, the appearance of abnormal
cell populations may also be correlated with the appearance of
other conditions, such as leukaemia, etc.
[0005] There are various conventional methods of cytological
analysis, involving microscopic examination after staining, and if
necessary after sedimentation or aggregation. The automatic
determination of blood cells began at the beginning of the 1960s
with the separation of the main normal leucocyte populations; see
the following bibliographic reference: (1) Hallerman L., Thom R.,
Gerhartz H. : "Elecktronische Differentialzhlzung von Granulocyten
and Lymphocyten nach intervaler Fluochromierung mit Acridinorange"
("Electronic Differential Counting of Granulocytes and Lymphocytes
by Interval Fluorochrome Staining with Acridine Orange"), Verh
Deutsch Ges Inn Med 70: 217, 1964.
[0006] The separation of the leucocytes was performed by flow
cytometry utilising various principles involving the optical and
chemical properties of the cells. Several automated haematology
analysers have been produced, using various techniques such as
Coulter's principle for determining volumes, measurement of
diffracted light for estimating sizes, measurement of diffused
light at 90.degree. for determining the internal structures of
cells, and fluorescence or absorption measurements for determining
the affinities of cells for various stains; see the following
bibliographic references 2 to 5:
[0007] (2) Adams L. R., Kamensky L. A.: "Fluorometric
Characterization of Six Classes of Human Leukocytes" Acta Cytol 18:
389, 1974;
[0008] (3) Shapiro H. M. et al. "Combined Blood Cell Counting and
Classification with Fluorochrome Stains and Flow Instrumentation"
J. Histochem Cytochem 24: 396-411, 1976;
[0009] (4) Terstappen L. W. et al. "Multidimensional Flow
Cytometric Blood Cell Differentiation Without Erythrocyte Lysis"
Blood Cells 17: 585-602, 1991;
[0010] (5) Terstappen L. W., Levin J. "Bone marrow cell
differential counts obtained by multidimensional flow cytometry"
Blood Cells 18 (2): 311-30, 1992.
[0011] The characterisation of cells in early stages of the cell
cycle has long been of interest to scientists and the
quantification of the RNA content of each cell has for a long time
been recognised as a representative parameter of this cycle; see
the bibliographic references 2 to 5 above and the following
bibliographic references 6 and 7: (6) Traganos F., Darzynkiewicz
Z., Sharpless T., Melamed M. R. "Simulataneous Staining of
Ribonucleic and Deoxyribonucleic Acids in Unfixed Cells Using
Acridine Orange in a Flow Cytofluorometric System" J. Histochem
Cytochem 25: 46, 1977; (7) Pollack A. et al. "Flow Cytometric
Analysis of RNA Content in Different Cell Populations Using Pyronin
Y and Methyl Green" Cytometry, vol. 3, no. 1, pages 28-35,
1982.
[0012] In their French patent no. 97 01090, dated Jan. 31, 1997 the
Applicants have already described a composition, and more
particularly a staining reagent, enabling this type of analysis to
be performed.
[0013] In order to automate such techniques various problems first
of all have to be solved, in particular reducing the treatment
times and cost of preparing the samples. Such a reduction may be
achieved in various ways, the most obvious being to reduce the
number of channels so that only one cell preparation is carried out
at any one time. This type of technique has previously been
described by Lon W. Terstappen (reference 4 above), but requires a
long treatment and analysis time, in particular for the accurate
counting of the nucleate cells, the number of which is often a
thousand times less than the number of erythrocytes.
[0014] In order to obviate this difficulty the biological sample is
often separated into at least two aliquot parts, one of which is
prepared at a certain concentration enabling the erythrocytes and
platelets to be studied, the other being prepared at a higher
concentration for the analysis of the nucleate cells.
[0015] These known techniques have various disadvantages.
[0016] Before the analysis, the treatment of this aliquot part
often involves the specific destruction of the erythrocytes in
order to facilitate the measurement of the remaining cells.
Although such a method enables the results of the measurements to
be obtained more quickly, this is nevertheless offset by the time
involved in the reaction, transfer and staining in order to obtain
the desired preparation.
[0017] The incubation time of a cell suspension in a reagent
solution is in particular associated with the time required for the
active principles to penetrate the interior of the cells. In French
patent no. 97 01090 mentioned hereinbefore, the Applicants have
described ways of accelerating this penetration involving the use
of an additive, in particular an ionophore type additive, to assist
the cell penetration.
[0018] The treatment time is also a function of the number of
successive stages that the aliquot part has to pass through. Lysis
and staining of the cells are often carried out in two successive
stages, in one order or the other (see U.S. Pat. No.
6,004,816).
[0019] These two dilution stages involve a not inconsiderable
expenditure in material, associated with a long minimum treatment
time.
BRIEF SUMMARY OF THE INVENTION
[0020] An object of the present invention is accordingly to provide
a reagent for the identification and counting of biological cells
that avoids the aforementioned disadvantages.
[0021] An object of the present invention is in particular to
provide such a reagent that enables the lysis of certain cells, in
particular erythrocytes, the fixing of the nucleate cells and the
staining of the intracellular material to be carried out
simultaneously.
[0022] An object of the present invention is also to provide such a
reagent that enables these operations to be carried out in a
conveniently short time in order to reduce to a large extent the
cost and time of the analysis and the number of reagents
involved.
[0023] The invention accordingly provides a reagent for the
identification and counting of biological cells in a sample, the
said reagent comprising:
[0024] a cell lysing agent selected from at least one detergent in
a concentration sufficient to lyse specifically a given type of
cell in the sample, and
[0025] a stain designed to mark the intracellular nucleic acids of
the remaining unlysed cell.
[0026] The invention thus provides a reagent for the simultaneous
lysing and staining of a biological sample, enabling a solution of
cells to be obtained in a single stage that can be analysed by for
example a flow cytometry system. This analysis enables the thus
treated cells to be classified and counted.
[0027] The reagent of the invention accordingly combines a reagent
solution of the type described in French patent no. 97 01090 with a
cell lysing agent that enables a given type of cells of the sample,
in particular erythrocytes, to be specifically lysed.
[0028] The staining reagent solution per se, described in French
patent no. 97 01090, enables the membrane permeation to be
accelerated for the subsequent staining of the biological cells of
the sample. This staining solution may be used before as well as
after the lysing of the erythrocytes, depending on the types of
cells being studied. The reagent principle of this staining
solution is thus preserved and introduced into a lysing solution,
enabling the erythrocytes to be destroyed and the remaining cells
to be stained before they are counted.
[0029] The cell lysing agent advantageously includes at least one
ionic and/or non-ionic detergent in a concentration capable of
lysing erythrocytes.
[0030] The detergent of the invention is advantageously selected
from:
[0031] primary amines, amine acetates and hydrochlorides,
quaternary ammonium salts, and trimethylethyl ammonium bromide;
[0032] amides of substituted diamines, diethanolaminopropylamine or
diethylaminopropylamide, amides of cyclised diethylenetriamine;
[0033] alkylaryl sulfonates, petroleum sulfonates, sulfonated
glycerides;
[0034] cholamides, sulfobetaines;
[0035] alkyl glycosides, saponins;
[0036] polyoxyethylene ethers and sorbitans, polyglycol ethers.
[0037] In one embodiment this detergent comprises a mixture of
Triton X100 in a concentration of 0.05% (w/v) and Tween 20 in a
concentration of 0.0001% (v/v).
[0038] Throughout the description the expression "w/v" denotes
"weight/volume" and the expression "v/v" denotes
"volume/volume".
[0039] The stain that is used is advantageously of the fluorescent
type.
[0040] Advantageously a stain is selected that is capable of
combining specifically with the intracellular ribonucleic acid and
enhancing its fluorescence once it has combined with the
latter.
[0041] The stain of the invention may be selected in particular
from the following stains:
[0042] thiazole orange or
1-methyl-4-[(3-methyl-2-(3H)-benzothiazolylidene-
)methyl]quinolinium p-tosylate,
[0043] thiazole blue,
[0044]
4-[(3-methyl-2-(3H)-benzothiazolyl-idene)methyl]-1-[3-(trimethylamm-
onium)propyl]quinolinium diiodide,
[0045] 3,3'-dimethyloxacarbocyanine iodide or
3-methyl-2-[3-(3-methyl-2(3H- )-benzothiazolylidene
-1-propenyl]benzoxazolium iodide,
[0046] thioflavine T,
[0047] the stains SYTO.RTM. and TOTO.RTM. (TM Molecular
Probes),
[0048] ethidium bromide,
[0049] propidium iodide,
[0050] acridine orange,
[0051] coriphosphine O,
[0052] auramine O,
[0053] the stains HOECHST 33258 and HOECHST 33342,
[0054] 4',6-diamino-2-phenylindole dihydrochloride (DAPI),
[0055] 4',6-(diimidazolin-2-yl)-2-phenylindole dihydrochloride
(DIPI),
[0056] 7-aminoactinomycin D,
[0057] actinomycin D, and
[0058] LDS 751.
[0059] In a preferred embodiment of the invention the reagent
moreover comprises at least one membrane penetration agent capable
of promoting the penetration of the stain into the cells to be
marked.
[0060] The agent promoting the membrane penetration is
advantageously an ionophore compound of the protonophore and/or
antibiotic type.
[0061] This agent is generally present in a concentration of less
than 0.005% (w/v). An example of an antibiotic that can be used is
valinomycin.
[0062] It is advantageous if the reagent moreover comprises at
least one membrane fixing agent present in a concentration of 0.1%
to 10% (w/v). This fixing agent preferably comprises at least one
alcohol and/or one aldehyde. Paraformaldehyde or glutaraldehyde for
example are preferably used for this purpose.
[0063] It is also possible within the scope of the invention to
include other additives or components in the reagent.
[0064] This reagent may accordingly also comprise at least one
compound selected from a complexing agent, an inorganic salt and a
buffer system.
[0065] According to another aspect, the invention relates to a
process for the identification and counting of biological cells in
a sample, in particular in a blood sample, which process comprises
the following operations:
[0066] mixing and incubating the sample with a reagent as defined
above in order to effect, in a single stage, the lysis of cells of
a given type, in particular erythrocytes, the staining of the
intracellular nucleic acids, and the fixing of the nucleate
cells;
[0067] measuring the resultant solution by flow cytometry using at
least two measuring parameters selected from resistive volume,
axial luminous diffraction, axial luminous transmission, orthogonal
luminous transfusion, and fluorescence; and
[0068] classifying and counting the nucleate cells in populations
by means of the measured parameters.
[0069] In the flow cytometry measurement the axial luminous
diffraction parameter is at least one parameter selected from small
angle diffraction and large angle diffraction.
[0070] This measurement may be carried out by means of a flow
cytometer having the conventional parameters such as axial
diffraction or "FSC" (forward scatter), orthogonal diffusion or
"SSC" (side scatter), either orthogonal fluorescence (FL1), axial
fluorescence or epi-fluorescence, all polarised or depolarised, as
well as additional measuring parameters such as transmitted light
measurement or resistive volume as described in French patent no.
89 14120 of Oct. 27, 1989.
[0071] The resistivity may be measured by means of a continuous
current in order to express the volume of the elements and/or by
means of a pulsed or alternating current in order to express the
internal densimetric differences approximating to the determination
of the structure.
[0072] These parameters may be used to obtain sets of
multi-parameter data for each of the analysed cells, enabling the
latter to be classified. The classification will be more precise
the more relevant and numerous the parameters defining the cells.
This type of multi-parameter study has already been described
before (see the bibliographic references 4 and 5 above).
[0073] Within the scope of the invention the classified nucleate
cells may either be mature or immature, or normal or abnormal
cells.
[0074] The classification of nucleate cells is carried out by known
processes. The classification may be performed by means of a
multidimensional analysis software program, with or without the use
of a software or other neuronal technique.
[0075] Within the scope of the invention the biological sample may
be a sample of a human or animal blood, or also a sample of
biological fluid or a suspension of cells of human or animal
origin.
[0076] This sample is mixed with the reagent solution under
specified temperature conditions. The reaction kinetics mean that
the erythrocytes are first of all destroyed, the penetration of the
stain being in parallel to the fixing of the cells, which takes
place more slowly.
DETAILED DESCRIPTION OF THE INVENTION
[0077] The invention will now be described by reference to the
following example:
EXAMPLE
[0078] Within the scope of this example a reagent is used having
the following composition:
1 Complexing agent EDTA 0.02% (w/v) Inorganic salt NaCl 0.85% (w/v)
Buffer system Phosphates 0.5% (w/v) Detergents Triton X100 0.05%
(w/v) Tween 20 0.0001% (v/v) Ionophore Valinomycin 0.003% (w/v)
Stain Thiazole orange 0.005% (w/v) Aldehyde Paraformaldehyde 1%
(w/v)
[0079] A sample of total blood is mixed with the above reagent
solution. After an incubation of a few seconds (typically of the
order of 15 to 30 seconds) the solution is analysed by means of a
flow cytometry system comprising at least the following parameters:
axial diffraction (FSC) providing an interpretation of the size,
orthogonal diffusion (SSC) expressing the structure of the elements
observed, and orthogonal fluorescence (FL1) enabling the expression
of the intracellular ribonucleic acid to be measured.
[0080] The results thus obtained are observed in multidimensional
mode so as to determine the interrelationships of the various
populations among each parameter.
[0081] Reference will now be made to FIGS. 1 to 4, which show the
results obtained with a sample of normal human blood.
[0082] FIG. 1 shows the matrix obtained by means of the two
parameters, axial diffraction (FSC) and orthogonal diffusion (SSC).
Four different populations can clearly be seen: L denotes
lymphocytes, M denotes monocytes, N denotes polynuclear
neutrophils, and E denotes polynuclear eosinophils. The populations
IG of immature granulocytes, BL of blastocytes, B of polynuclear
basophils and ErB of erythroblasts are shown but cannot be
differentiated in only two dimensions.
[0083] FIG. 2 shows the matrix formed by the axial diffraction
(FSC) and fluorescence (FLI) parameters. The same four populations
as shown in FIG. 1 can be seen, but arranged differently. The
mononucleate cells L and M form the upper group of average
fluorescence, and the polymorphonucleate cells N, E and B form the
lower group of weak fluorescence. The population ErB of
erythroblasts is clearly separated at the apex of the two groups
thus formed. The normal positions of the populations BL and IG are
shown.
[0084] FIG. 3 shows the matrix formed by the orthogonal diffusion
(SSC) and fluorescence (FL1) parameters. The same populations are
found organised in a different way, but enabling the populations IG
and BL to be isolated (in very small amounts in a normal
sample).
[0085] FIG. 4 shows a three-dimensional representation of the
populations obtained.
[0086] FIGS. 5 and 8 show the same types of results as FIGS. 1 to 4
respectively, but obtained with a sample containing blast cells
(B1) and treated according to the invention.
[0087] FIGS. 9 to 12 show the same types of results as FIGS. 1 to 4
respectively, but obtained with a sample containing immature
granulocytes (IG) and treated according to the invention.
[0088] The reagent and the process of the invention thus enable a
specific lysis and a simultaneous staining of biological cells in a
sample, in particular in a sample of human or animal blood, to be
carried out in a single stage.
[0089] Cells may thus be identified and counted rapidly using an
automated analysis system based on flow cytometry.
* * * * *