FIELD OF THE INVENTION

The present invention relates to the analysis of cellular parameters in biological samples using indirect immunofluorescent techniques. More particularly, the present invention is directed to the fluorescent detection of nonviable cells in assays involving whole blood lysis and fixed cell suspensions using flow cytometric methods.

BACKGROUND OF THE INVENTION AND RELATED ART

Blood is composed of two main parts: (1) plasma, the fluid portion, which consists primarily of water in which are dissolved proteins and many inorganic and organic substances carried by the blood to and from the tissues; and (2) blood cells, the particles suspended in the plasma, making up about 45 percent of total blood volume and including erythrocytes (red blood cells) , leukocytes (white blood cells) , and thrombocytes (platelets) .

The leukocytes are the body's primary defense against infection. In healthy individuals, there are 5,000-10,000 leukocytes per cubic millimeter of blood, and they consist of three types: (1) granulocytes (neutrophils, eosinophils, and basophils) , which can

phagocytose bacteria;

(2) monocytes, which phagocytose cellular debris and interact with lymphocytes in the processing of antigens in the immune reaction; and (3) lymphocytes. Lymphocyte population in blood is defined by a number of subclasses which play distinct roles in the immune response. For example, the relative number of lymphocytes in various subclasses is likely to change in disease states. Hence, enumeration and identification of cells of the various subclasses yields an indication not only of the constituency of the blood in particular, but generally with respect to the relative well being of the organism.

There are two principal classes of lymphocytes concerned with the immune response. B lymphocytes are bone marrow-derived lymphocytes that migrate to the tissues without passing through or being influenced by the thymus. These cells play a major role in humoral immunity; on stimulation by antigen, they mature into plasma cells that synthesize humoral antibody. T lymphocytes are lymphocytes that either pass through the thymus or are influenced by it on their way to the tissues. T lymphocytes can suppress or assist the stimulation of antibody production in B lymphocytes in the presence of antigen, and can kill such cells as tumor and transplant tissue cells. Particular subclasses of functionally distinct lymphocytes can be distinguished on the basis of antigenic determinants on the cell surface.

The ability to identify or suppress classes or subclasses of T lymphocytes is important for diagnosis or treatment of various immunoregulatory disorders or conditions. For example, certain leukemias and lymphomas have differing prognosis depending on whether they are of B cell or T cell origin. Thus, evaluation of the disease prognosis depends upon distinguishing between these two classes of lymphocytes. Certain disease states, e.g., juvenile rheumatoid arthritis and certain leukemias, are associated with an imbalance of T cell subclasses. It has been suggested that autoimmune diseases generally are associated with an excess of helper T cells or a deficiency of certain suppressor T cells, while malignancies generally are associated with an excess of suppressor T cells. In certain leukemias, excess T cells are produced in an arrested stage of development. Diagnosis may thus depend on the ability to detect this imbalance or excess. In renal allograft recipients, monitoring of T cell subsets in peripheral blood provides information which can be used as a basis for clinical decisions. Since significance is attached to relatively small changes in the sizes of the subpopulations of T cells, it is necessary to have an accurate, reproducible method for obtaining data regarding the T cell subpopulation. Thus, the detection and identification of cell types in the hematopoietic system is a useful research and clinical tool.

Recently, monoclonal antibody techniques have been utilized to produce large quantities of highly purified antibodies to various lymphocyte subclasses. Utilizing such antibodies, it has proven feasible to assay an individual•s lymphocytes to determine the relative numbers of cells in various subclasses. Further, utilizing direct or indirect techniques, the antibodies may be fluorescently tagged, thereby rendering the samples under consideration amenable to flow cytometric analysis. Staining the cells with colored or fluorescent dyes enhances the visibility of cells and subcellular components such as chromosomes and makes possible the characterization and measurement of cellular parameters, particularly cell surface antigen detection for cell type and subset analysis.

In recent years, the availability of multiparameter flow cytometers has made it possible for large numbers of cell samples to be rapidly and quantitatively analyzed by direct and indirect immunofluorescence. Flow cytometry is generally accepted as a tool to aid in the identification of or discrimination between cell types and between various functional and/or maturational subsets within a cell type. In flow cytometry, cells are dispersed in fluid suspension and flow one at a time through a narrow beam of light, typically from an argon laser. Each cell generates optical signals that are measured and analyzed. The signals can be scattered light, which relates to the mass of the cell, and

fluorescence, which relates to the amounts and molecular environments of the dyes used to stain the cell. Flow cytometers are described in more detail in Herzenberg et al . (Scientific American , Vol. 234, pp. 108-117, 1976) and in US 3,826,364, US 4284,412, and US 4,661,913.

Conventional immunofluorescence techniques presently include the physical separation of the lymphocytes from other leukocytes and the erythrocytes as a preliminary step, usually by density gradient centrifugation. This separation step eliminates the possibility that non- lymphocyte cells, i.e., erythrocytes, monocytes and granulocytes might be counted as specifically stained lymphocytes. This initial lymphocyte isolation step is long and arduous; in fact, this step is much longer than the relatively simple steps of tagging and analyzing the tagged lymphocytes. The necessity of separating the lymphocytes from other leukocytes and erythrocytes is a serious impediment to rapid clinical analyses. Furthermore, even for research applications, where a fast analysis time is less critical, the lymphocyte separation step involves the risk of loss of some lymphocytes, which introduces uncertainty and inaccuracy into the subsequent analysis.

More recently, whole blood lysing techniques are being used which avoid the necessity for prior separation of lymphocytes from other blood cells. Typically, a sample of whole blood is selectively tagged so that a select subclass of leukocytes is provided with a

distinguishing marker. The tagging is preferably accomplished by incubating the sample with an antibody which is selectively reactive with a distinct antigenic determinant on the surface of cells of the select subgroup. The antibody is typically conjugated to a fluorochrome which provides it with a predetermined fluorescence which responds to a given optical stimulation. The erythrocytes are then lysed so as to break the erythrocytes into fragments. The sample, which contains the leukocyte population of which a select subclass has been tagged, is then passed, substantially a cell at a time, through an area of focused optical stimulation so as to determine the cells which have been tagged with the antibody while detecting light scattered by and emitted from the cells. The cells of the selected subclass are thus differentiated from other cells based at least in part on occurrence of the predetermined fluorescence response to the optical stimulation.

Unfortunately, a serious drawback to whole blood lysis techniques is the nonspecific staining of damaged and nonviable cells by some antibodies. This nonspecific uptake of fluorochrome-conjugated antibodies by dead cells and cell debris, i.e., artifactual staining, cannot be distinguished from that which is specific. Several means have been used in an attempt to distinguish intact or viable cells from nonviable cells. Otten and Loken (Cytometry, Vol. 3, pp. 182-187, 1982) distinguished viable thymocytes from nonviable thymocytes on the basis

of their forward angle light-scattering characteristics due to the loss of membrane integrity. This technique is limited, however, in that it can only be used with cell populations that are homogeneous with respect to their cell size and light-scattering characteristics.

Certain dyes can be used to distinguish intact cells from damaged or nonviable cells. Propidium iodide, for example, will penetrate and stain nonviable cells but not intact cells. However, staining with propidium iodide is reversible, and propidium iodide may leak out of dead cells and intercalate with the DNA of previously viable cells whose membranes become permeabilized by the fixation or lytic processes. Such uncontrolled uptake may, in turn, alter the evaluation of viable (non- propidium iodide stained) cells.

Sch id et al . (Cytometry, Vol. 13, pp. 204-208, 1992, hereinafter Schmid) used 7-aminoactinomycin D (7- AAD) in the place of propidium iodide as a fluorescent nonvital DNA dye for discriminating nonviable cells from viable cells. Schmid, however, failed to appreciate nor attempted to solve the problem of distinguishing cells that are nonviable prior to cell fixation and/or lysis.

In US 5,057,413, Terstappen et al . (hereinafter Terstappen) utilize the DNA dye LDS-751 to discriminate intact from damaged cells following fixation. As with the other methods using a DNA intercalator, however, Terstappen's method still does not permit distinguishing cells that were nonviable prior to lysis and/or fixation

steps from those that are nonviable following lysis and/or fixation.

Riedy et al . (Cytometry, Vol. 12, pp. 133-139, 1991, hereinafter Riedy) used ethidium monoazide to distinguish nonviable from viable cells in fixed specimens. Ethidium monoazide binds irreversibly to cells with damaged membranes by photochemical crosslinking with nucleic acids in the cell, and thus it is unable to leak out of cells like propidium iodide. In Riedy's method, the cells were lysed before being treated with ethidium monoazide. Unfortunately, ethidium monoazide photoactivation is only 15% efficient, and much of it is washed out of the cells during fixation and washing. Thus, the stained cells are much less bright than cells stained by propidium iodide. Moreover, the photolabelling must be performed separately from staining, thereby increasing the analysis time. This presents a serious drawback to the routine use of the procedure. Accordingly, it is an object of the present invention to provide a method for detecting and discriminating nonviable cells in a sample before and following steps that per eabilize or damage cell membranes. It is a further object of the present invention to provide a method for identifying and excluding from analysis cells that are nonviable at the time of immunofluorescent staining.

Yet a further object of the present invention is to provide a test kit for performing the method of the present invention.

As used herein, "intact cells" means cells which have not been treated with fixatives or lysing agents and which do not show obvious morphologic damage or functional impairment. "Nonviable cells" refers to those cells that are dead, damaged, not intact, or whose membranes have become permeabilized through lysis or fixation steps.

SUMMARY OF THE INVENTION

The present invention comprises a method for fluorescent detection and discrimination of nonviable cells in a sample by the use of a complementary pair of nucleic acid dyes, thereby permitting the differentiation of binding by the nucleic acid dyes by nonviable cells before and following steps that permeabilize or damage cellular membranes, e.g., lysis and fixation steps. The nucleic acid dyes are preferably DNA dyes and are selected such that their DNA binding characteristics are essentially the same, i.e., both dyes bind to common binding sites on DNA, but such that the dyes differ in their fluorescent emission characteristics. An especially preferred complementary pair of dyes is that of 7-aminoactinomycin D (7-AAD) and actinomycin D (AD) . 7-AAD is a dye excitable at 488 nm and therefore suitable

for use in flow cytometers with argon-ion laser excitation, with fluorescence emission resolvable from the standard immunofluorescent fluorochromes such as fluorescein isothiocyanate (FITC) and phycoerythrin (PE) . When subjected to optical stimulation such as an argon laser, 7-AAD emits a red fluorescence at greater than 630 nm. On the other hand, the DNA dye AD exhibits no red fluorescence emission when subjected to the same optical stimulation. In a particularly preferred embodiment of the present invention, a whole blood or tissue sample containing unlysed cells is first contacted with the immunofluorescent stain of choice, i.e., an antibody to the cellular antigen of interest which has been conjugated to a fluorescent label. The labeled antibody is allowed to bind to the cells of interest, and there may also be some nonspecific binding by the antibody to nonviable cells. Following or at the time of the addition of the immunofluorescent stain, cells in the sample are also contacted with a first member of a complementary dye pair, preferably 7-AAD, which binds to the nucleic acid in the nonviable cells. After washing the cells to remove excess antibody and to minimize residual 7-AAD, a molar excess of a second member of the complementary dye pair, preferable AD is added. Lysis and/or fixation steps are then performed in the customary manner as desired. The AD binds to the DNA of the cells

made permeable during lysis and fixation, and the presence of a molar excess of AD competitively inhibits the uptake of residual 7-AAD by the newly permeable cells, thereby making it possible to distinguish cells that were nonviable before lysis from those that were nonviable after lysis.

The invention further comprises a test kit comprising a labeled antibody selective for a particular cellular marker or constituent and capable of emitting fluorescence upon stimulation. The test kit further comprises a complementary dye pair, one of which members has a measurable fluorescent emission distinguishable from that of the antibody label.

DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing competitive inhibition of 7-AAD and AD binding at 7-AAD concentrations ranging from 2 μg to 20 μg.

Figure 2 is a contour plot produced by two color flow cytometry apparatus illustrating various features of the invention. Plotted on the Y-axis is green fluorescence (LIGRF) from cells stained with fluorescein labeled anti-thy-1.2. Cells simultaneously stained with 7-AAD emitted a red fluorescence which is plotted on the X-axis (IRFL) .

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises a method for fluorescent detection and discrimination of nonviable cells in a sample by the use of a complementary pair of nucleic acid dyes, thereby permitting the differentiation of binding by the nucleic acid dyes by nonviable cells before and following steps that permeabilize or damage cellular membranes, e.g., lysis and fixation steps. The nucleic acid dyes are preferably DNA dyes and are selected such that their DNA binding characteristics are essentially the same, i.e., both dyes bind to common binding sites on DNA, but such that the dyes differ in their fluorescent emission characteristics. An especially preferred complementary pair of dyes is that of 7-aminoactinomycin D (7-AAD) and actinomycin D (AD) . 7-AAD is a dye excitable at 488 nm and therefore suitable for use in flow cytometers with argon-ion laser excitation, with fluorescence emission resolvable from the standard immunofluorescent fluorochromes such as fluorescein isothiocyanate (FITC) and phycoerythrin (PE) . When subjected to optical stimulation such as an argon laser, 7-AAD emits a red fluorescence at greater than 630 nm. On the other hand, the DNA dye AD exhibits no red fluorescence emission when subjected to the same optical stimulation.

In a particularly preferred embodiment of the present invention, a whole blood or tissue sample

containing unlysed cells is first contacted with the immunofluorescent stain of choice, i.e., an antibody to the cellular antigen of interest which has been conjugated to a fluorescent label. The labeled antibody is allowed to bind to the cells of interest, and there may also be some nonspecific binding by the antibody to nonviable cells. Following or at the time of the addition of the immunofluorescent stain, cells in the sample are also contacted with a first member of a complementary dye pair, preferably 7-AAD, which binds to the nucleic acid in the nonviable cells. After washing the cells to remove excess antibody and to minimize residual 7-AAD, a molar excess of a second member of the complementary dye pair, preferable AD is added. Lysis and/or fixation steps are then performed in the customary manner as desired. The AD binds to the DNA of the cells made permeable during lysis and fixation, and the presence of a molar excess of AD competitively inhibits the uptake of residual 7-AAD by the newly permeable cells, thereby making it possible to distinguish cells that were nonviable before lysis from those that were nonviable after lysis.

The invention further comprises a test kit comprising a labeled antibody selective for a particular cell surface marker and capable of emitting fluorescence upon stimulation and further comprising a complementary dye pair, one of which members has a measurable

fluorescent emission distinguishable from that of the antibody label.

EXAMPLE I

Competitive Inhibition of 7-AAD and AD Binding

A 6-8 week old BALB/c mouse was sacrificed by cervical dislocation and the thymus was removed by dissection. The thymus gland was rinsed with 70% ethanol to remove any erythrocytes and was placed in a 15 ml conical centrifuge tube containing 10 ml cell culture medium RPMI (Gibco) supplemented with 10% fetal bovine serum. The cells were counted using a hemacytometer and diluted with phosphate buffered saline (PBS) containing 0.1% sodium azide and 0.1% bovine serum albumin to 1 x 10 ⁶ cells per ml. One ml of cells was placed into each of 32 12 x 75 mm borosilicate sample tubes. The tubes were centrifuged at 200 x g for 10 minutes at 4° C. The supernatant was discarded, the cell pellet was resuspended, and 0.5 ml of a 1% paraformaldehyde solution was added to each sample tube. The samples were then incubated for 30 minutes at 4°, following which 0.5 ml of PBS was added to each tube, and the samples were then centrifuged at 200 x g for 10 minutes at 4° C. The previous washing step was repeated two times using 1 ml of PBS. The cells were resuspended in the following dye solutions prepared in 0.1 % TRITON X-100 (TM, Rohm &

Haas, Philadelphia, PA) . The total volume of each sample was 0.5 ml.

Sample AD Cone. AD Volume 7-AAD 7- -AAD Vol. PBS ! Volume μg 2. 5 mg/ml Cone. μg 1 mg/ml

1 500 200 20 20 280

2 250 100 20 20 380

3 100 40 20 20 440

4 50 20 20 20 460

5 25 10 20 20 470

6 12.5 5 20 20 475

7 0 0 20 20 480

Sample AD Cone. AD Volume 7-AAD 7- -AAD Vol. PBSi Volume μg 2. 5 mg/ml Cone. μg 0 .5 mg/ml

8 500 200 10 20 280

9 250 100 10 20 380

10 100 40 10 20 440

11 50 20 10 20 460

12 25 10 10 20 470

13 12.5 5 10 20 475

14 0 0 10 20 480

Sample AD Cone. AD Volume 7-AAD 7- -AAD Vol. PBS ; Volume μg 2. 5 mg/ml Cone. μg 0. .25 mg/ml

15 500 200 5 20 280

16 250 100 5 20 380

17 100 40 5 20 440

18 50 20 5 20 460

19 25 10 5 20 470

20 12.5 5 5 20 475

21 0 0 5 20 480

Sample AD Cone. AD Volume 7-AAD 7-AAD Vol. PBS '■ Volume μg 2.5 mg/ml Cone. μg 1 mg/ml

22 500 200 2 20 280

23 250 100 2 20 380

24 100 40 2 20 440

25 50 20 2 20 460

26 25 10 2 20 470

27 12.5 5 2 20 475

28 0 0 2 20 480

Sample AD Cone. AD Volume 7-AAD 7-AAD PBS Volume μg 2.5 mg/ml Cone. μg Volume

29 0 0 0 0 450

30 0 0 0 0 500

31 0 0 0 0 500

32 500 200 0 0 300

Note: Sample #29 had 100 μl of 0.5 mg/ml propidium iodide added. The AD and propidium iodide used were from Boehringer Mannheim, and the 7-AAD was from Sigma.

The samples were incubated for 30 minutes at 4°C. Following incubation, 0.5 ml of PBS was added to each sample and they were centrifuged at 200 x g for 10 minutes at 4°C. The supernatant was discarded, the cell pellet was resuspended and the previous wash step was repeated two times using 1 ml of PBS. The samples were then resuspended in 0.5 ml of PBS. The samples were analyzed on a Coulter EPICS Profile II flow cytometer with 15 milliwatt laser emission at 488 nm. Data generated is given in the following table:

Sample 7-AAD AD Mean tration μg Fluorescence

1 20 500 65

2 20 250 77

3 20 100 123

4 20 50 173

5 20 25 228

6 20 12.5 284

7 20 0 431

8 10 500 26

9 10 250 32

10 10 100 53

11 10 50 76

12 10 25 109

13 10 12.5 139

14 10 0 272

15 5 500 16

16 5 250 19

17 5 100 27

18 5 50 35

19 5 25 58

20 5 12.5 72

21 5 0 197

22 1 500 14

23 1 250 14

24 1 100 16

25 1 50 20

26 1 25 25

27 1 12.5 29

28 1 0 107

Example II

Flow Cytometric Detection of Nonviable Cells

Human lymphocyte preparation:

Human peripheral venous blood was collected by venipuncture into VACUTAINER (TM, Becton Dickinson) blood collection tubes. The whole blood (8-10 ml) was diluted to 30 ml with phosphate buffered saline (PBS) containing 0.1% sodium azide and 0.1% bovine serum albumin (BSA). The diluted blood was then slowly poured over a 10 ml layer of lymphocyte separation medium (Boehringer Mannheim) in a 50 ml conical polypropylene centrifuge

tube. The tube containing two liquid layers, the separation medium and diluted blood, was centrifuged at 400 x g for 30 minutes at 20°C. After centrifugation, the buffy coat layer (second layer from top) was collected by pipetting and placed in a 15 ml conical polypropylene centrifuge tube. This layer was diluted with 5 ml of PBS and centrifuged at 400 x g for 10 minutes at 20°C. The supernatant was discarded and the cell pellet was resuspended in 8 ml of PBS. The mixture was centrifuged at 400 x g for 10 minutes at 20°C. The supernatant was discarded and the cell pellet was resuspended in 8 ml of PBS containing 0.25% TRITON X-100. The lymphocytes were killed by alternating 15 minute incubations at 56°C and at -20°C until the cells were 100% dead by Trypan Blue (Gibco) exclusion. The cells were counted using a hemacytometer.

Mouse thymocyte preparation:

A 6-8 week old BALB/c mouse was sacrificed by cervical dislocation and the thymus was removed by dissection. The thymus gland was rinsed with 70% ethanol to remove any erythrocytes and was placed in a 15 ml conical centrifuge tube containing 10 ml cell culture medium RPMI (Gibco) supplemented with 10% fetal bovine serum. The cells were counted using a hemacytometer.

Detection of cells:

Human and mouse cells were mixed together in 12 x 75 mm borosilicate culture tubes according to the table below:

Sample Mouse Thymus Human Percent of Total

Lymphocytes

1 1 X 10° cells 5,000 cells 0.5

2 1 X 10 ⁶ cells 10,000 cells 1

3 1 X 10 ⁶ cells 20,000 cells 2

4 1 X 10 ⁶ cells 50,000 cells 5

5 1 X 10 ⁶ cells 100,000 cells 9

6 1 X 10 ⁶ cells 200,000 cells 17

7 1 X 10 ⁶ cells 500,000 cells 33

8 1 X 10 ⁶ cells 1,000,000 cells 50

9 1 X 10 ⁶ cells 1,000,000 cells 50

10 1 X 10 ⁶ cells none 0

11 1 X 10 ⁶ cells none 0

12 none 1 x 10 ⁶ cells 100

13 1 X 10 ⁶ cells none 0

Next 0.5 ml of PBS was added to each sample tube and the samples were centrifuged at 200 x g for 10 minutes at 4°C. The supernatant was discarded and the cell pellet was resuspended. The appropriate FITC-labeled monoclonal antibody (Boehringer Mannheim) was added to each sample tube according to the table below:

Sample Antibody Concentration

1 Anti-thy-1.2-FITC 1 μg

2 Anti-thy-1. 2-FITC 1 μg

3 Anti-thy-1. 2-FITC 1 μg

4 Anti-thy-1.2-FITC 1 μg

5 Anti-thy-1.2-FITC 1 μg

6 Anti-thy-1.2-FITC 1 μg

7 Anti-thy-1.2-FITC 1 μg

8 Rat anti-DNP 11-FITC 1 μg

9 Anti-thy-1.2-FITC 1 μg

10 Rat anti-DNP 11-FITC 1 μg

11 Anti-thy-1.2-FITC 1 μg

12 Anti-thy-1.2-FITC 1 μg

13 Anti-thy-1.2-FITC 1 μg

Then 20 μg of 7-AAD (Sigma) was added to each sample tube except #13. The samples were incubated in ice for 30 minutes in the dark. Then 1 ml of PBS was added to each sample and the samples were centrifuged at 200 x g for 10 minutes at 4°C. The supernatant was discarded and the cell pellet was resuspended. The two previous steps were repeated two times for a total of three washes. At the end of the third wash, the samples were resuspended in 0.5 ml of a 1% paraformaldehyde solution. Next 50 μg of AD (Boehringer Mannheim) was added to each sample and the samples were incubated for 30 minutes on ice in the dark. Then 0.5 ml of PBS was added to each sample and the samples were centrifuged at 200 x g for 10 minutes at 4°C. The supernatant was discarded and the cell pellet was resuspended. The two previous steps were repeated two times for a total of three washes. After the third wash, the samples were resuspended in 0.5 ml PBS for flow cytometric analysis. The samples were analyzed on a Coulter EPICS Profile II flow cytometer using a 15 milliwatt helium neon laser emitting at 488 nm. Figure 2

displays the results for sample #9 (50% live/50% dead cells) .

A kit comprising containers separately containing a labeled antibody, such as a fluorescein labeled antibody to a cell surface marker, and a pair of complementary dyes such as the preferred 7-AAD and AD is disclosed and may be conveniently used in the practice of the present invention. It will be appreciated by those skilled in the art that the antibodies may be labelled before inclusion in the kit or separate containers containing the fluorochromes may be included for independent labelling.

Those skilled in the art will appreciate that other methods of cell preparation and other lysis and fixation techniques other than those described in the examples set forth herein may be substituted without departing from the inventive concepts of the instant invention. Good discussions of cell preparation, fixation methods, staining techniques, dyes and markers, and can be found in "Practical Flow Cytometry", 2nd Edition, by Howard M. Shapiro (Alan R. Liss, Inc., 1988) and in "Flow Cytometry and Sorting", 2nd Edition, M.R. Melamed et al., Eds. (Wiley-Liss, Inc., 1990). All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains.

It will be apparent to one of ordinary skill in the art that a number of changes and modifications can be

made in the invention without departing from the spirit or scope of the appended claims. For instance, it will be appreciated by those skilled in the art that a fluorescence microscope may be substituted for the automated instrument. In this case, manual counting and identification of cells is required.