CA1296622C - Method and apparatus for automated assessment of the immunoregulatory status of the mononuclear leukocyte immune system - Google Patents

Abstract

METHOD AND APPARATUS FOR AUTOMATED ASSESSMENT OF THE
IMMUNOREGULATORY STATUS OF THE MONONUCLEAR LEUKOCYTE
IMMUNE SYSTEM
ABSTRACT OF THE INVENTION
The ability of mononuclear leukocytes to respond to standard stimuli is measured based on the expression of activation antigens on mononuclear cell subclasses. In a preferred embodiment, a sample of mononuclear leukocytes is cultured for up to 24 hours with a standard stimulus known to activate such cells. After culturing, aliquots of the cells are incubated with fluorophore-conjugated monoclonal antibodies to antigenic determinants of a particular mononuclear subclass and different fluorophore-conjugated monoclonal antibodies to particular activation antigens. The incubated aliquots are analyzed on a flow cytofluorometer, whereby each cell is illuminated with a particular light (e.g. argon ion laser), which detects and measures forward light scatter, orthogonal light scatter and two different wavelengths of light emitted from the fluorophores. These parameters are used to identify and enumerate the cells of different subclasses present within the mononuclear leukocyte sample, the cells of said subclasses which have been induced to express a particular activation antigen and the quantity of the activation antigen on said cells. An analysis of these enumerations is shown to correlate with the immunoregulatory status of the mononuclear leukocyte immune system. Data generation and analysis can be performed using a flow cytofluorometric apparatus with data and control signal processing to ensure accuracy and reproducibility of the results of the assay.

Description

METH(:3D AND APPARATUS ~OR AUTOMATED ASSESSMENT OE' THE
IMMUNOREGULATORY STATUS OF TEIE MONONUCLEAR LEUKOCYTE
IMMUNE SYSTEM
FIELD OF THE INVENTION
s Thi~ invention relates to an apparatus and automated method to perform an analysis of the mononuclear ~: leukocyte ;mmune system's ability to re pond to standard ~; stimuli. Data concerning activation antigen expression on particular subclasses of stimulated mononuclear cells that have interacted over time is collected using flow cytofluorometric techniques. An analysi~ o~ activation ~; antig~n expression on these stimulatQd mononuclear cell subclasses correlates with the immunoregulatory ~tatus of the mononuclear leukocyte immune 5y~tem~
T~i~ a~ay provides an analysis of the ; interaction of mononuclear cell~ in vitro, such interaction dependent u~on the immunoregulatory status of the individual from whom the sample o~ aells was obtainedO
Therefore, the re~ult~ of thi~ A~say can b~ used as a measur~ of the immunoregulatory ~tatus of ~he in y~Q
mononuclear leukocyte immune ~ys~em. Analysis o~ this data can explain the heterogeneity o mito~en response among normals, better define immunoregula~ory abnormalitie~ i~ a variety of immune-mediated disorders, and provide a m~thod for monitoring in vivo immunomodulation therapy involving ~uppression or potentiation o mononuclear cell activation. The assay ; also involves the use of a 10w cytofluorometric apparatus having certain modifica~ions, in terms of da~a and control '. ' , , ' .

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: signal proce sing, which allows for more accurate and reproducible measurements of the stimulated mononuclear c~lls .

BACKGROUND AND PRIOR ART
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The immune system is a regulatory system tha~
maintains homeostasis by protecting the body against forPign particles, such as pathogenic microbial agents, and against native cells that have u~dergone neoplastic transformation. The immune system exerts its control withln the body by virtue of circulating components, ~:' humoral and cellular, capable o acting at sites removed from their point o~ origin. The complexity of the immune ; system is derived from an intricate communications ne~work capable of exerting multiple e~fects basQd on relatively distinct cell typ~s.
:~ The cellular component of the immune sy~tem co~sists o~ relatively distinct cell types. Usiny morphologic criteria the cellular component can be divided into cla~ses; e,g. granulocyte~, lymphocytes and monocytes, The morphologic criteria include differences in cell size and intracellular organelles such as the nucleu~. Of these clas~es, lymphocytes, monocytes and ~:: related cells are grouped together as mononuclear cells.
Further distinction of cell types involves dividing the cells into subcIasses using certain cell surface structures termed antigenic determlnants; that is, within a class of cells there exist particular antigenic 3~6 --3~

determinants which define relatively distinct cell types, i.e., subclasses. It ~hould be noted that other antigenic determinants or combination~ of antigenic determinants can be used to further delineate subclasses; the ~erm class, hereinafter, refers to distinction of cell types based on morphologic criteria, while ~ubclass refers to the distinction based on criteria relating to morphoIogy and expression of antigenic determinants.
The major mononuclear cell classes of the immune system are monocytes and lymphocytes. There exists other classes of ~ononuclear cells; e.g. lymphoblasts and large : granular lymphocytes thought to be natural killer cells.
Peripheral blood monocyte~ are derived from bone marrow monocytes. Human lymphocytes are derived from two areas, the thymu~ and the "bur3a"-equivale~t. Thes~ areas are ~ referred to as the central lymphoid sy~tem. The : peripheral lymphoid sy~tem consists of matur~ lymphocytes which can be ~ound in lymphatics, spleen, lymph node~, - lymphoid tissue of the GI tract, respiratory tract~, sectory glands and blood. Mononuolear leukoaytes found at the~e "peripheral" locations are hereinafter re~erre~ to as peripheral mononuclear cell~. Mature mon~nuclear leukocytes are referred to as immunocompetent cell3 and taken`,together constitute the mononuclear leukocyte immune system.
:~ Mononuclear leukocytes can be divided into subclasses. For example, there ar~ two major types of lymphocytes; T lymphocyte~, which are referred to as T-cells and B lymphocytes which are referred to as B-cells.
T-cells constitute a group of related subclasses which ,. .
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participate in a variety o~ cell-mediated immune re~ctions. These subclasses can be distinguished in a ~ample of leukocytes by the~r morphology which identifies ~hem as lymphocytes and the particular antige~ic determinants which delineate the subclass. T-cell subclasses are involved in different cell-mediate regulatory functions, such as enhancement or suppression of an immune response, and are directly involved in effector functions, such as the cytotoxic destruction of viral infectedr foreign or mali~nant cells.
~` Mononuclear cell~ communicate, i.e. interac~, by direct cell-cell contact or via soluble factors. These soluble ~actors are termed lymphokine~, if secre~ed by lymphocyte~l or monokines, if secreted by monocytes.
Immunocompetent cells can expr2ss highly ~peci~ic receptors for a particular lympho/monokine on their cell ~urEace. Binding o~ a lympho/monokirle with its receptor : initiate~ or facllitateq certain in~racellular events.
; For example, the proliferation of lymphocyte~ i~
:: 20 in~luenced by certain lymphokines. Interleukin~2 ~h~reinafter IL~ a lymphocyte derived factor that promote~ long-term proliferation o~ T-cell lines in culture~ Upon activation, the T-c~ll produces IL-2 and IL-2 receptor~, al50 referred to as IL2R~ The binding of IL-2 to IL--2 rec~ptor~ triggers T-cells to proceed from the Go/Gl into the S phase of the cell cycle (D.A. Cantrell and K.A. Smikh, "Transient ~xpression of Interleukin-2 Receptorso Consequences for T-C*ll Growth,"
:~ Journal Qf Experimental~Medicine~ (15~ 95~1911 (1983)) and regulakes the production of other lymphokines. The `:

:~ failure of production of either IL-2 or its receptor, ~esults in failure of many T-cell immune response~. XL-2 : receptor~ have al~o been found on B-cell~ and monocyte.s.
IL-2 also upregulates the e~pression of IL-2 receptors, i.e. it increases ~he amount of IL-2 receptors expressed on the cell surface. K. Welte, et al.f "In~erleukin 2 Regulates The Expression of TAC Antigen on Peripheral Blood T Lymphocytes," Jo~rnal of Expe~ri~ental Medicine, ~160) 1390-1403 ~1984]- An example of an important monokine is interleukin-l (hereinafter IL-l) which appears : to be essentlal for the amplification o~ many T-cell dependent immune responsesO IL-l induces the expression of certain E-rose~te receptor~ and ~timulat~s the : production of the lymphokine, IL-2. S.B. Mi:zelJ
"Interleukin-l and T cell Activatî~n," XmmunQlQqic Review, ~63) 51-72 (1982). It is believed tha~ a particular 44 kilodalton protein~ found on 80~ of mature T-cells; might ~unction as the receptor ~or the monokine, IL~l~
The induction of the mononuclear l~ukocyte immune 9y5tem respon~e to a foreign ~e.g. viru~ or organ transplant) or altered ~e.g. neop}a tlc) antig~n lnvolves the activation of the mo~onuclear cells/ ~uch as lymphocytes, with r~ceptors ~or the particul~r antig~n.
~hi~ activation entails a se~uence Qf event~ initiated when the antigen binds to the receptorO Activation provides for cell differentiation and proliferation into a clone of cells to respond to the an~igen.
Activation i~ not necessarily a linear sequence of events, often events occur simultaneously, and not all ;, 30 events lead directly to cell division~ Important activation events include: cross-linking of certain cell surface molecules, certain intracellular events leading to actlvation of certain enzymes, such enzymes facilitating ~: increased protein synthesis including productio~ of certain lympho/monokines and activation antigens, expression of activation antigens including certain lympho/monokine receptors on the cell surface, ~he regulation o~ these events by enhancing or suppressing regulatory signals (e.g. certain lympho/monokines), replication of D~A and cell division. The increase in protein synthesis can occur as early a~ one hour, with DNA
replication beginning about 36 hours, after the initial stimulus. These activation event~ are not n~cessarily uniform for each mononuclear cell subcla~æ and the response to the regulatory signal~ i8 not uniform;
therefore, th~ activation of the cells of the mononuclear leukocyte immune system i~ a3ynchrsnous in nature due ~o their heterogeneity. R.F. A~hman, "Lymphocyte Ac~ivation", ~ , 267-300 (198~)~
A stimulus can initiate mononuclear cell activation by cro~ linking certain cell ~r~ace moleaules. An antigen achieves ~uch cro~-linking when it i9 rendered functionally multivalent after in~eraction with antigen-pre~enting cells, o~ten monocytes. Certain multivalent glycoproteins, termed lectins, obtained from :~ plant or animal sources can cro~s~ k certain surface ~: molecules thereby activating lymphocyt~s D some even induce division of cells. Substances which initiate activation leading to division of cells are termed mito~ens.

Examples of lectins, which act as mitogens, include phytohemasglutinin (hereinafter PHA), concanavalin A and pokeweed mito~en.
On lymphocytes, both the T-cell antigen receptor S and the E-rosette receptor surface molecule~ can be crosslinked thereby activating cells. For example, the monoclonal antibody OKT3 which identifies and binds to the T-cell antigen recep~or can act as a mi~ogen. The : mitogenic effects of the lectin phytohemagglutinin are mediated via the E-rose~te receptor. Therefore, stimuli which initiate activation of the mononuclear leukocyte immune system can include a~tigens, an~ibodies and certain lectins. Multivalency~ which is important ~or cro~s-linkinq, is either an inh~rent property of th~ activating sub~tance, achieved biologically by interaction with antigen-presenting cells or achieved artificially by binding to a subs~rate, e.g. Sepharo~e beads.
After a ~timulus has initia~ed a ~equence of activation events, there is an increaset exp~ession o~
certain cell ~urface proteins not ~asily detected on re~ting cells. These are re~erred to as activation antigens. Activation antigens include receptor~ for cer~ain lympho/monokines. One such activa~ion antigen is . the IL-2 receptor~ which is also referred to as ~he T~C
antigen/ and has been shown ~o be de~ec~able within 6 hours and reaches maximal expr~ssion 72 hsurs after initiation of activation~ D.A. Cantrell and K.A. Smith, : ~.

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~: Other activation ~ntigens expressed on the cell `~ membrane include DR, the transferrin receptor, an epi~ope ~; of the E-rosette receptor termed T113, Tl~, Tal, Ba, 4F2, 3 and Act I. See, e.g., A.I. Lazaroutis, et al., S "~ymphocyte Activation Antigens: I. Monoclonal Antibody, Anti-Act I, Defines a New Late Lymphocyte hctivation ~ Antigen," Journal_of Immunolo~, (133) 1857-62 ~1984~.
:~ Particular activation antigens are no~ necessarily unique to one class or subclass of mononuclear cells and they are not each expressed at the same time during the sequence of ~:, ac~ivation events. Therefore, at a certain length of time after a stimulus has initiated mononuclear cell . activation, the degree to which a c~rtain activation antigen is expressed will depend on the particular :1 15 sequence of preceding events and the regulatory in~luences on the mononuclear cell which expres6~s it.
The function o~ all the activation antigens is ~:~ not yet known. The transferrin receptor bind~ transf0rrin which has been shown to enhance response of mo~onuclear cell~ to mitogens and might be involved in natural killer cell differenti~tion. The expre~sion of receptor~ ~or cer~ain lympho/monokine~, e.g> IL-2 rec~p~or, is important in the propagation of activation event~ a~t~r initiation by a stimulus.
' The re~ponse of th mononuclear leukocyte immune system to foreign or altPred antigens, thus involves a ~; complicated regulatory network consisting of difer~nt . subclasses of cells with distinct functions interacting by direc~ contact and/or lympho/monokines. Damle~ et al., ~: 30 "Immunoregulatory T Cell Circuits in Man," Journal of ':

: I~munoloqy, ~134~ 235-43 (1985). A particular stimulus will initiate a sequence of activation event~ in individual cells of certain mononuclear cell subclasses.
The sequence i9 not necessarily linear~ nor do all cells begin or ~pend an equal amount of time at each step of the sequence. The sequence of activation events includes expre~sion of receptors for and production of certain lympho/monokines each with particular regula~ory functions ~ with regards to mononuclear cell differentiation and :~l lO proliferation. J.J. Farrar and W.R. Benjamin, "The Lymphokine Cascade: a Systemic Model of Immunore~ulation,"
Requlation of the mmune Re~onse, 8th Int~ Convoc.
Immunol., 76-87 (1982).
Variations in the sequence of activa~ion event~
1 15 are dqtermined by the subclass to which the cell belongs and th~ e~ect~ o these regulatory signal~. Thi~
activation of individual cells of the mononuclear leukocyte immune system in the pre~ence o the system's regulatory communications network rQ~ults in dif~erentiation and clonal proliferation o~ regulatory and effector 3ubclas~es, neutralization of the inciting : factor(~) and eventual return o~ the ~y#t~m to a ~teady ~tate, which can be more or les~ quiescentO This state determines the mononuclear leukocy~e immune syst~m's ability to respond to a stimulu~ appropriately with regards to the length of time ~o initiate, the nature and the intensity of the response. This "initial" stat~ of the mononuclear leukocyte immune system can be termed ~he immunoregulatory status.
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Over the pa~t 10 years t ne~ methodologies have been developed to analyze the complex regulatory and effector functions of the mononuclear leukocyte immune system. The development of monoclonal antibodies ha~
facilitated the identification and enumeration of the different subclasses in the mononuclear leukocyte immune ~ystem. These antibodie3 can bind to cell surface structures, e.g. antigenic determinants. Th~ binding of a monoclonal antibody to a cell can be determined using a fluorescent microscope, if the antibody has been directly or indirectly conjugated to a fluorophoreO Monoclonal ~ antibodiss to the T-cell antigen receptor, e.g. anti-OKT3, : anti-CCT3 and anti-Leu-4/ are used to identify and ! ::
enumerate T-cells~ Monoclonal antibodies can be used to `~ 15 identify and snumerate other mononuclear cell subclasses, : for example: helper/inducer ~ lymphocytes, e.g. anti~OKT4, anti-CCT4 and anti-Leu-3a; ~uppressor/cytotoxic T
~: lymphocytes, e.g. anti OKT5, anti-OKT8, anti~CCT8 an~
anti-Leu~2a; natural killer cells, e.g. anti-Leu~
cell3, e.g. anti-Leu-12; monocy~es, e.g. antl~Leu-M3.
Kung in U.S. Pat~nt 4,364,932 and in UuS. Patent 4,381~932 and i~ U.S~ pat~nt 4,381,245; GrE~ M~nti, et al., "~ormal ;~ ~uman Blood Den~ity Gradient Lymphocyt~ Subset Analysis:
I. An ~nterlaboratory Flo~ Cytometric Comparison of 85 Normal~Adults," American Journal of ~ematoloqy (2) 41-52 ~: (1985); 2tc~
ntibodie3 binding to the same or similar antigenic de~erminants are u~ed ~o yroup these determinants into "Clusters of Dif~erentiation."
30 A. Bernard, et al., "The Clusters vf Differentiation (CD~

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Defined by the First International Workshop on Human Leucocyte Differentiation Antigens," Human Immunoloqy, (ll) l-lO ~l984). It ~hould be noted that the antigenic determinants identified by monoclonal antibodies can be found on cells from diff~rent classes of mononuclear leukocytes9 e.g. Leu-3a is found on monocytes and lymphocytes. As stated previously~ subclass di~tinction : relies on both morphologic and antigenic determinant ~ criteria. Also, the amount of antigenic determinant ; lO expressed on the cell surface and identified by monoclonal .:antibodies can change with activation of the cell, ~.g.
the amount of Leu-4 appear~ to d~crease a~ter initiation of the activation.
Further subclass delineation often requires lS identifying two distinct antigenic determinants on the same cell. For example, the inducer of ~uppre~ion ~ubclass can be identified and enumerated u~ing anti-Leu-3a and anti-Leu-8 or anti-2H4 r the helper ~ubcla~s can be identifi~d and enum~rated u~ing anti-Leu-3a and anti-4~4, and the ~uppres30r sl1bcla~s can be identified an~
enumeratcd u~ing anti-Leu-2a and anti-Leu-l5. N.K. Damle, ibld~, ~tc.
Monoclonal antibodies have al50 been de~loped which bind to the activation antigen~, e.gO anti-O~T9 (trans~errin r~ceptor~ and anti-Interleukin-2 Recep~or (hereinaft~r anti-IL2R)0 To Uchiyama, et alO, 11~
Monoclonal Antibody (Anti Tac) Reactive with Activated and Functionally Mature ~uman T-Cells~" Journal of Immunolo~y (126) 1393-1403 (1981); D.L. Urdal, et al., "Purification and Chemical Characteri2ation of the Receptor for : ~ .

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Interleukin-2 from Activated T Lymphocyte~ and from a ~uman T-cell Lymphoma Cell Line", Proc. Natl. Acad. Sci., (81) 64~1-85 ~19~4).
Flow cytofluorometric technigues have been developed to differentiat~ mononuclear oell ~ubclasses, such a~ helper/inducer and suppressor/cytotoxic T-cell~, u3ing monoclonal antibodies and an apparatus to determine morphologi~ characteristics and to detect different antigenic determinants. ~ansen in ~.S. Patent 4,2B4,412 discusses a method and apparatus for automated identification and ~numeration of ~pecified blood cell lymphocyte subclasses. In ~ansen'~ assay, a ~ample of :~ whole blood or buffy coat i9 incubated with a monoclonal ~:~ antibody which i~ selectively reactiv~ with a di tinct ~: 15 antigenic determinant identifying a .~ubclas~ of lymphocyte~. Antibodie~ which bind to a particular antigenic determinant are conjugated to a ~uorophore ~alao referred to as a fluorochrome or fluorescer), dir~ctly or indirectly, such that they will b~
fluorescently re~ponsive to particular light (ePg. argon ion la~r). 5ingle cells are iden~ified and differentiat~ based on the mea~urem~nt o~ two light scatter parameters and the amount of fluore~cence. ~anges of value~ are set using electronics or the two light ~catter paramet~rs such that ~7an ar~a of interest", al50 : referred to as a "window", is formed discriminating the ~; lymphocytes. For each CQl1 in the area of interest, the amount of fluorescence is used to determine if ~he cell ~ has the antigenic determinant expression typical of the :~ 30 subclass.

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It should be noted that in addition to whole blood or bufy coat, appropriate ~amples for incubation can be obtained from the interface after density gradient centrif~gation of whole blood. Marti, et al., ibid. This interface yields mo~tly mono~uclear cells, although it can contain immature granulocy~es. ~lso, the samples of incubated cells can be fixed with 1~ paraformaldehyde and stored at 4C for approximately seven days until : performing flow cytofluorometric measurements.
A flow cyto~luorometer is an instrument capable :~ of measuring properties of single cells as they pass ~; through an orifice at high velocity. I~ S~h~r ~ M7 Mage, ;~ "Cellular Identification and Separation," F~ndamentals : Immunology, 767-68 (1984). The measuremen~s made using a flow cytofluoromet~r are performed on cells ~u~pended in a a ~tream of fluid that i~ intersected by a beam o~ light, e.g. coherent light from an argon ion las~r. Lasers ar~
particularly importan~ in these sy~tems ~ince khey provide a ~ource of intense, highly collimated ~p~rallel light waves~ and monochromatic light, which can b~ ~ocused to deliver a large amount of energy to the cells being analyz~d. When a cell in th~ fluid stream inter~ects the beam, the light i9 scattered. Low angle forward (approximat~ly 2-15 degree~ to the angle of incident light~ and orthogonal (approximately 90 degrees ~o the intersection of the incident light and stream) scattered light can be detected and measured. It should be noted ~ that light scatter at other angles can also be detected : and measur~d. The low angle forward (hereinater forward) ~ 30 light scatter detector iB located direct~y in line with qJ~

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the laser beam. The orthoyonal light scatter and ~; fluorescent collection lens is placed orthogonal to the intersectio~ o~ the laser beam and stream. Measurement of orthogonal light scatter utilizes a photomultiplier tube (hereinafter PMT). The light accepted by the forward : light ~catter detector and orthogonal csllection lens i5 approximately a cone of half angle 20.
The measurements of forward and orthogonal light scatter have been found to relate to cell size and intracellular structures, respectively. It hould be no~d that certain flow cyto1uorometer~ use a measurement of céll volume, instead of forward light ~catter, as one ~: o~ two parameters to delineate leukocyte c}a~sesO Red blood cells, platelets and debris yield ~he lowe~t levels ~: 15 of forward and orthogonal light scatter. Thxeshold triggers on the flow cytofluorometer can be set ~o that light scatter data will not be generated by theseO Dead ~ c8115 will yield lower forward and orthogonal light : scatter mea~urements than their live counterparts. Due to di~erences in size and intracellular structures, dif~erent leukocyte clas ~ can be differentiated using the forward and orthogonal light scatter parameters.
Han~en, ibidD; R.~. Hoffman, et al. t "Simpl~ and Rapid ~:~ Measur~ment of ~uman T Lymphocytes and Their Subcl~sses in -: 25 Peripheral Blood,~' Proc. Nat'lO AcadO Sci., ~77~ 1914-17 (1980~; ~offman in U.S~ Patent 4~492,752. Most lymphocytes will yield a low level of both forward and :~ orthogonal light scatter. ~ost monocytes will yield a higher level of forward and slightly higher level of orthogonal light scatter. It should be noted that most ~15-granulocytes will yield a level of forward light scatter overlapping that of lymphocytes and monocytes~ however~
the orthogonal light ~catter will be much higher than either of the~e cIasses. Light scatter "areas of int~rest" can be demarcated such that each of theQe classes can be identified. There are important mononuclear cell classes which overlap the~e divisions based on Eorward and orthogonal light scatter parameters.
: These include lymphoblasts nd natural killer cells, which : 10 can yield light scatter parameters similar to monocytes.
~ In ~uch cases fluorophore-conjugated monoclonal antibodies ; can be u~ed to facilitate the delineation of ~ubclasses of these mononuclear cell classes.
The la~er i5 often tuned so that light of 488 nanometers (her2inafter nm3 is produced. Thi~ wavel~ngth o light provide~ ~n optimal ~xcitation ~or 1uorescein-isothiocyanate (hereinafter ~ITC)~ FITC is commonly used a~ a ~luoroptlor~ which i~ conjugated to monoclonal an~ibody probes. When the cell being analyzed ha~ bound ~0 ~luorophore-conjugated rnonoclonal antibody~ light is ~mitted from the excitat.ion of ~h~ fluorophore by the laser liqhty e.~. FITC emits light in ~he green spectrum (herei~after green fluorescence). The ligh~ emitted enter~ a collection lens and then passes through a series of filters, whi~h allow only a certain wavelength of light to enter a fluorescence detector, usually a PMTo Fluorescence detectors, as well as li~ht scatter detectors, produce an electronic signal as output which can be used by a device to analyz2 the measured parameter data.

The amount of fluore~cence emitt~d by a cell in the laser beam is proportional to the number of fluorophores excited. A monoclonal antibody can be ~:conjugated to a known number of 1uorophores. Therefore~
~:~S the ~ize of the electroni~ signal produced by the fluorescent detector is proportional to the number of monoclonal antibodies bound to the cell and thereby i5 a measure of the number o~ antigenic determinant~ (or activation antigens) detected on the cell surface.
~10 Therefore, a cell ~an be assi~ned to a specific subclass :wi~hin a class of mononuclear leukocytes using forward light scatter, orthogonal light scatter and fluorescence parameters.
With a flow cytofluorometer confiyured with .15 three PM~s ~et along an axis orthogonal to the stream and the light beam axes, two wavel~ngths of emitted light can ~;be detected in addition to orthogonal light sc~tter.
Filters are used to isolate the wavelength o~ emitted :light to be detected by each PMT. This technlque :20 facilitates dual parameter fluore C~Rt measurement~ of cells which have been incubated with two monoclonal antibodies, each bindi~g to a differ~nt antigeni~
structure on the cell surface, e.g. two different antigenic determinants. The monoclonal antibodies are u~uall~ directly conjugat~d to fluorometrically distinguishable fluorophores which emit different wavelengths of light when illuminated with laser ligh~D
~ven wi~h the use of fil~ers to isolate specific wavelengths of emitted light for detection, there is usually some overlap of the e~is ion spectra detec~ed by the PMTs. Mo~t flow cytofluorometers have a~ electronic compensation network which can be set by the operator to correct for the overlap. M.R. Loken, et al., "Two~Color Immunofluorescence Using a Fluorescence-ActiYated C~ll Sorter," Journal of ~istochemistry~and Cytochemistry, (25) 899-907 (lg77).
In addition to FITC, Texas Red dye (conjugated to avidin and then incubated with a biotinylated monoclonal antibody) is often used for dual fluore~cent measurements. This dye is optimally ex~ited by light at a wavelength of S68 nm thereby requlring a second laser~
The light from the second la~er i~ focused o~to the ~tream : : lightly below the intersect: point of the first laser.
Such a two laser system requires appropriate signal delay electronics ~o that light scatter and both ~luorescence parameters are as3e~ed ~imultaneouslyO ~ recently :~ discovered ~luorescent compound, phycoerythrin (hereinafter PE) (~ee Stryer in U,S. Patent 4~520,110), can b~ conjugated ~o monoclonal antibodie~ and utilized ~0 for dual parameter fluoresGent measurements with FITC by a ~ingle la~er sy~tem, as it can al~o be excited by light at a wavelength oE 488 nm. The liyht emitted by PE is in ~he orange-red spectrum (hereinafter red fluorescence), V.T.
Oi, et,al., '~Fluor~scent Phycobilipro~ein Conjugates for ~nalyq~ of Cells and Molecules," Journal.of Cell Biolo~y, f93) 981-86 (1982). The use of du~l parameter fluorescent ~ measurement~ has facili~ated further subclass delineation : within classes of mononuclear leukocyte~ and ha~ clariEied antigenic determinant expression on subclasses, e.g. high level~ of Leu-2a are found on the suppressor/cytotoxic ~ubclass while lower levels of Leu-2a ar~ found on natural : killer cells. L.L, Lanier and M.R~ Loken, "~uman Lymphocyte Subpopulations Identified by Using Three-color Immunofluorescence and Flow Cytometric Analy~i~," Journal of Immunolo~, (132) 151-156 (~984).
To facili~ate analysis, data generated by a flow : cytofluorometer is usually displayed as a frequency distribution plot, i.e. histoyram, with one or two measured parameter~ displayed. Each two parameter histogram resembles a topographic map with the contours ~; depicting number of cells. The histogram can di~play data :~` for each cell analyzed or only for ~ho~e with parameter data within an area of interest. The amount of antigenic determinants and activa~ion antigen~ detected per cell ha~
lS a large range necessitating the use of a loga~ithmic scale to repre~ent fluorescenc~ parameter data for all the cells on th~ histogram. Thi~ is ofte~ accomplished u~ing a logarithmic amplificat.ion o the electronic slgnal ~: produc~d by the preampliier in the PMT ~this ~ignal i~
; 20 o~ten lntegrated so as to be proportional to the total ~luore~cence detected by the PMT~. It has been found that many antigenic determinants have a lognormal distribu~ion thereby facilitating analysi~. This lognormal di~tribution i~ often found even after ntigenic ~5 determinant modulation due to cell activation. ~naly~is of hi~tograms representing flow cytofluorometr1c data has been used to enumerate subclasses of mononuclear cells in an attempt to assess the immunoregulatory status of the : mononuclear leukocyte immune system.

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An a}tered immunoregulatory status is su~pected to be involved in many human di~ease~, e.g. viral illnesses, autoimmune disease and malignancy. Autoimmune disease involves a primary defect in the mononuclear : 5 leukocyte immune system such that self-antigens can initiate activation of the mononuclear cells. In the case of organ transplantation, suppression o~ the normal îmmunoregulatory status with medication i5 required to avert rejection of the transplanted organ.
Immunosuppression, whether secondary to medication or due to a disease state, i~ suspected to contribute to the development of certain malignancies. I. Penn, "D~pressed ~: Immunity and the Development o~ Cancer t ~I Clinical and Experimental Immunolo~y~ (461 453-474 (19Bl~. Disor~ers o normal human phy~iologic functions involvi~g the immune system can be referr~d to as immune-mediated disorders.
Peripheral ~lood samples have been analyz~d using monoclonal antibodies and flow cytofluorometry to determine the percentages o~ T-cell subclasses in patient~
with di~fer~nt immune-mediated disorders. M.A. Bach and J.~. ~ach, "The Use o Monoclo~al Anti-T Cell Antibodi~
to Study T~Cell Imbalances in Human Disea es," ~1lL~_~Z
~mmunol., (45) 44g-456 (1981). For example~ an increase in the ratio of helper/inducer to suppres~or/cyto~oxic (her~inafter ~:S~ T-cells have been found in many patients with autoimmune di~eases or ~ultiple ~clerosis. Increased numbers of suppressor/cytotoxic T-cells have been found in patients with viral illnesses, giving rise to a decreased H:S ratio. In patients with the acquired immunodeficiency : 30 syndrom~ (hereinafter AIDS~, there is also a decr2ase in ol~

~2~-the H:S ratio, however thi is du~ to decreases in the number of helper/inducer T-cells. In patie~ts with ~olid tumors, a decrease in the number of helper/inducer and : suppressor/cytotoxic T-cells is found, al~hough the ; 5 p~rcen~ages were normalO These abnormali~ies in T-cell subclasses have not been found con~istently in all patients with certain diseases and often the abnormalities do not correlate with the degree of di~ease activity. For example, the H:S ratio has not been found to correlate with disease activity in patients with systemic lupus erythematosus. J.S. Smolen, et al., "Heterogeneity of Immunoregulatory T-Cell Subsets in Systemic ~upus ~ Erythematous: Correlation with Clinical Features,"
;:~ American Journal of Medicine, (72~ 783-790 (1982), Also, patient~ lnfe~ted with HTLV III, th0 virus thought to be She etiology o~ AIDS, dg not nece~arily have d~creased : nurnber of helpex/inducer T-c~
Ia _tro, mononuclear leukocyte as~ays have been developed in an attempt to mea~ur~ the ability o~ the mononuclear leukocyte immune ~y~t~m ~o unction appropriately. Traditional in y~p ~unctional ~s~ay~ u~e : mitogens to induce blasteogene3i~. The amoun~ of blasteo~ene~ then measured by tritiated thymidine uptake in replicating DNAI Thi~ type of assay i5 performed using a fixed concentration oE i~olated mononuclear cell~, which are cultured in the presence sf ~ an optimal concentration of a mitogen and then pulsed with : tritiated thymidine. Tritiated thymidine uptake is generally measured after culturing the cells for 72 hours.
These ass~ys, however~ do not allow for an analysis of the mononuclear cell subcla~ses which have interacted during the induction of blasteogenesis. B~F. Hayne~, et al.
"Immune Response of ~uman Lymphocytes In vitrQ," ~r~g In Clinical Immunols~y, (4~ 23~62 (19803, Certain diseases are as~ociated with decreased (e.g. malignancies or AIDS) or increasea (e.g. multiple sclerosis) in Y~ responses to mitogens used in these : assays. Immunosuppressive agents, e.g. glucocorticoids and cyclo~porin, have been shown to suppress blastogenesis. Research involving immunosuppressive ayents has revealed differential inhibitory effects on certain subclasses of lymphocytes. ~hese traditional :~ mitogen a says, how~ver~ do not allow for an analysis o the lymphocyte subclasses which have interacted during ~he ~: 15 induction o~ blastogenesis or or measurement of the differential inhibitory or potentiatlng ef~ects which contribute to an alt~red immunoregulatory status~ F/
Kristensen, et al., "Human Lymphocyte Proliferation: ~.
. Cor~elation between T-l~mphocyte~ n~ iLQEL~æ~~
(5) 5~-63 (1982).
S~milarlyl mea~uring IL-Z receptor expre~ion on the mononuclear cell cla~se~, e.g. lymphocyte~, without subclass distinction also has not proven to be useful in delin~ting many cf the possible etiologies o an altered immunoregulatory statu~. Activation of T-cell~ results in the expression of specific cell surface receptors for :~ lymphokinès, e.g. IL-2 receptor, and the synthesis of :~ lymphokines, e.g. IL-2. Immunosuppressive agents, e,g.
glucocorticoids and cyclosporin, have been ~ound to inhiblt pro~ein synthesis and specifically to block .
' production of IL-2 by mon~ucLear cells stimulated with mitogens.
Studies using the monoclonal antibody, anti~TAC, indicate that cyclosporin does not block the expression of IL-2 receptor in such cells. T. Miyawaki, et al., "Cyclosporin A Does Not Prevent ~ Expression of Tac Antigen, a Probable TCGF Receptor Molecule, on ; Mitogen-Stimuluted Human T-Cells," Journal of Immunology, (13) 2737-42 (1983). In patients infected with HTLV III, only certain patient groups were found to not express IL-2 receptor after stimulation with mitogens. Patients wi-th SLE have normal expres-sion of IL-2 receptor after stimulation with mitogens, while those with rheumatoid arthritis have decreased expression of IL 2 recep-tor. N. Miryasaka, et al., "Interleukin 2 Deficiencies and Systemic Arthritis and System Lupus Erythematosus," Clin. Immuno.
Immunopath., (31) 109-17 (1984)o Cereborspinal fluid lymphocytes from MS patients have normal proportions of IL-2 receptor bearing cells, although they had deficient production oE IL-2.
SUMMARY OF THE PRESENT lNVENTIOM
One aspect oE the present invention provides a method for assessing the immunoregulatory status oE the immune systern by generating and analyzing data on activated ceL]s in select mono~
nuclear cell subclasses Erom a sample oE mononuclear cells cuLtured with a standard stimulus comprising:
f / isolating a sample which is substantially comprised of peripheral mononuclear cells;
culturing the sample of mononuclear cells with a standard stimulus for a period of -time sufficient to aIlow for measurable cellular activation, as influenced by cell subclass interaction, to develop;
.:
~,~

generating data on individual cells of the sample indicative oE select mononuclear cell subclasses and cellular activation;
performing a first analysis oE the data to identi:Ey and enumerate cell.s in the select mononuclear cell subclasses;
performing a second analysis of the data to iden-tify and enumerate activated cells in the select mononuclear cell sub-classes; and performing a comparison of the enumerations of the first and second analyses to determine the degree of cellular activa-tion in the select mononuclear cell subclasses, the degree being an assessment of the immunoregulatory status of the immune system~
Another aspect of the present inven-tion provides an apparatus useful for performing the method. The apparatus comprises:
flow means for passing cells of the sample rapidly and substan-tially one at a time through a sensing zone; means for stimulating fluorescent activity of first and second fluorophores passing through the senslng zone;
photosensing means for detecting light scatter from the sensing zone in at least one predeterm:ined direction lnd:icati.ve o~
cell morphology; photosensing means :Eor sensing fluorescence from the first fluorophore in the sensing zone, indicative of -the select cell subclass;
photosensing means for sensing fluorescence from the second fluorophore in the sensing zone, indicative of the activation antigen; and means connected to the photosensing means for determining the ..
.
~! .

~13~:~$~
-~ 23a ~

quantity oE cells in the select cell subclass and the quantity of cells expressing greater than a preset minimal density of activation antigen.
The present invention relates to a method and apparatus to assess the immunoregulatory status of the mononuclear immune system. The assay described herein distinguishes the altered immunoregulatory status of patients with immune-mediated disorders or receiving immunomodulation therapy from the immunoregulatory status of normal individuals. The method involves culturing mono~
nuclear cells with a standard stimulus and measuring the quantity o~ activated cells in particular mononuclear cell subclasses. An analysis of these measurements is used to determine the degree of cellular activation in the mononuclear cell subclasses, i.e. -the ability of the mononuclear immune system to respond to stimuli~
The degree of cellular activation is used to assess the ln _vo immunoregulatory status of -the mononuclear immune system.
The assay afEords a timely (oEten less than about 2~
hours), reproducible and accurate assessment oE the immunoregula-tory status. These attributes are necessary for a clinical assay to assist the diagnostic and therapeutic decision proc~ss, e.g.
monitoring for transplanted organ rejection where delay oE ade-quate immunosuppresslve therepy could result in loss of an organ.
In particular, this invention relates to a method where-by a sample consisting substantially of peripheral mononuclear cells is cultured with a standard stimulus for a period of time sufficient to initiate a sequence of activation events and allow for measurable cellular ac-tivation, as influenced by subclass `~

~2~;6~
~ ~ 23h ~

interaction, to develop. The assay does not involve isolation of specific subclasses prior to stimulati.on, and thereEore prese~~ves and measures the enhancing and suppressing effects o other sub-class components of the system as they~are present ln vivo.
Standard stimulus, Eor the purposes of this assay, is deined as a stimulus consisting of a substance(s) at a kno~n concentration(s) such that a reproducible measure of particular activation ~s antigen expression on sel~ct ubclass(es) can be obtained on repeat determinations from a ample of mononuclear cells from a normal individual, each after ~he same l~ngth of time in culture with the gtimulu5. Examples of : 5 3tandard stimuli include, but are not limited to:
mitogenic lectins, Sepharose-bound anti-OKT3 with IL-l and Sepharo~e-bound speciic antigen with IL-l and IL-2.
Measurement of the amount of cell surface activation antigen expression on select subclasses is performed using fluorometrically distinguishable fluorophore-conjugated monoclonal antibodies and a flow cytofllloromet~r to collect light scatt~r and flu~rescence parameter data for each cell. The light ~atter data i5 used to identify a particular leukocyt~ class, while fluorescence data i5 analyxed to e~umerate the cell ~ subclass and the amount of activation antigen expre~sion.
`~ The quantlty of activated cell~ in particular 7ubclasses can be determined using a preset minimal amount or a pre~et range of activation. The degree of cellular ackivation in select mononuclear cell ~ubclasse~ i~
determined ~rom the analys~ of this flow cyto~luorometric data and u~ed to a~sess the immunoregulatory ~atus.
Generation and analysis of data concerniny leukocyte clas~, cell subcla3s and thP amount of activation antigen 25 expreg5ion i5 performed using a flow cytofluorometric apparatus including a computer dev ce with appropriate software programming. This system contain~ data and :~ control signal processing to ensure the accuracy and reproducibility of the mea~uremen~s made and therefore the 30 results produced by the apparatus.

~5-In particular, the present invention can be u ed to determine thP ability of T lymphocyte subclasses to respond t~ standard stimuli in vitro, while preserving their ability to interact, as they do in ~Q. The assay can enumerate certain T-cell subclasses by detecting cells bearing certain antigeniG determinants on their cell membrane, e.g. Leu-3a or Leu 2a. After culturing ~ lymphocytes for lB hours with the standard stimulus, PHAd :: these T cell subclasses express a measurable amount of the ~ 10 activation anti~en, IL-2 receptor. An analysis to :~ determine the deyree of activation of these T-cell ~` subclasse~ gives a clinical measure of ~he immunoregulatory status o~ the individual. In a similar ~ashion, the assay can determ~n~ the degree of activatisn of other mononuclear c~ ubclas~eæ~ thereby re~ining this clinical mea~ure.
Usiny the3e technique~ and procedure~ the present invention provides a method and apparatus which i8 ~ufficiently 3ensitive and speci~ic to asse~s the in ~Q
kinetics of altered self-regulation of, and e~ec~s of immunomodulation therapy on, the mononuclear leukocyte immune system.

: BRIEF DESCRIPTION OF T~E DRAWINGS

The his~rograms shown in thase figures were generated from the four parameter data measured by an : EPICS V flow cytofluorometer using the MDADS software package ICoulter Corporation, Hialeah, FL). Each two parameter histogram has the x and y axes labeled for the .~ .

~.,,, ~ '. , , 3~

para~eters they representO Each axis i~ divided into 64 equally spaced divisions referred to as channel~. The orward light ~catter (FLS) axis is linear~ The orthogonal (LI90), green fluoresoence (LGF) and ed flurosc~nce tL~) axes are logarithmic. The Z axis relates to the number of cell5 and is linear. In these two dimensional plots, contours are used to indicate Z-axis values and are represented by a scatter density plot.
Below each two parameter histogram are the thr~e levels at which oontours are drawn. For each one parameter histogram, the y-axis relates to the number of cells. The scale factor for the y-a~is is in the upper le~t-hand corner. The total number o~ cells represent~d in the histogram is in the upper rlght-hand corner.
In some of the figur~, areas o~ interest are demarcated with geometric ~hapes or curs~r lin~s~ The x and y coordinates of the point~ u~ed to ~en~rate the geoMetric shape3 are to the left of th~ hi~togram~ When u~ed, there are four cursor lines for each two parameter histogram and two ~or each one parameter histogram. The cha~nel numbers at which the cur~or lines are drawn is indicate~ below each histogram~ For two parameter : hi~tograms, the axis label ~e.g. LGF~ is followed by two ;~ number~, each indicating a channel a~ which a cursor line is drawn. For one parameter histograms~ the word "C~ANNEL" is followed by two numbers indicating wherP the two cursor }in~s are drawn. Also, the number of cells with data parameters which would place them within the specified geometric shape or between the cursor lines as .
:;

~, . . .

drawn i~ indicated after the word "INTEGAL"~ This is expressed as a percent of the total cells represented in ~ ~he hi~togram after the words "~ I~ INT~R~A~".
::~ FIG. 1 shows a histogram of forward light :~ S scatter and orthogonal light scatter data a~ measured by a flow cytofluorometer of leukocytes i801ated by d~nsity ~: gradient centrifugation of peripheral blood with areas of interest demarcated by solid lines.
FIG. 2 shows a histogram of forward light scatter and orthogonal ligh~ scatter data as measured by a flow cytofluorometer of the same leukocytes as in FIGo 1 with one area of interest demarcated.
FIG. 3 ~hows unanalyæed two parameter light : scatter data as ~easured by a flow cyto~luorometer of : 15 leukocytes isolated from a periphexal blood sample by ~;~ density gradient centri~ugatio~ a~ter culturing with P~
for 18 hours.
FIG. 4 shows th~ ~ame data for the sam0 leukocytes a~ in FIG. 3 with an area of interest indicative o~ lymphocyte~ demarcated, -- FIG. 5 ~hows ~nan~lyz~d two p~ramet~r fluore~cence data o~ ~timlllated mononuclear cells incubated with both anti-IL2X-PE and anti-Leu-3a~FITC and determin~d to be lymphocyte~ as in ~IG, 4.
, FIG. 6 ~hows a hi~togram o~ green fluorescence data con tructed using only tho~e cells in the zero and ~ one red fluore~cent channels from FIG. S.
:~ FIG~ 7 shows the same data for the ~ame ~:~ leukocytes as in FIG. 5 with a cursor line ~t at ~he approximate x-intercept a~ determined in FIG. 6.
;:

:

~28-FIG. 8 shows the same data for the same leukocytes as in FIG. 7 with a second cursor set at the red fluorescence channel 10.
: FIG~ 9 ~hows the same data fvr the ~ame S leukocytes as in FIG. 7 with a thîrd cursor ~et at the red fluorescence ~hannel 25.
FIG. 10 shows ~wo parameter ~luorescence data oE
: stimulated mononuclear cells incub~ted with both anti-IL2R-PE and anti-Leu-2a~FITC and determined to lymphocytes ~imilarly to FIG. 4. With cur~ors set in a similar manner as in FIG. 8.
FIG. 11 shows unanalyæed two parameter ~ fluorescence data of ~timulated mononucl~ar cell~
; incubated with both mouse IgGl-PE and mouse-IgGl-FITC and determined to be lymphocyte~ ~imilarly to FIG. 4.
FIG. 12 shows a histogram ~imilar to FIG~ 9l FIG. 12A shows a histogram similar to ~IGI 9.
FIG. 13 show~ a one p~rameter histogram of red fluore~cence data ~logarithmi~ ~cale) a~ mea~ured by a ~low cyto~luorometer of stimulated mononuclear c~115 incubated with antl-IL2R-PE and determined to be lymphocyte~ ~imilarly to FIG. 4.
FIG. 14 shows a sch~matic diagram of a preerred embodiment ~or the apparatus including flow cytofluorometric and signal processihg hardware~ a ~; microcomputer system and software to ~acilitate clinical assessment of the immunoregulatory status of th~
mononuclear leukocyte immune 3ystem.

- ~9 -:~: DET~ILED DESCRIPTION
While this invention is satisf ied by embodiments in many different forms, there is shown in the drawing~
and will herein be described in detail a preferred embodiment of the invention, with this underst~nding that the present disclosure is to be considered exemplary of the princip}es of the invention and is not intended to ~: limit the invention to the embodiment illu~trated. The examples which follow this detailed description are offered by way of further illustration and not by way of : limitation. The s~ope of the invention will be measured : by the appended claims and their equiYalents.
~ In the pr2sent assay, a sample containing --~ substantially p~ripheral mononucl~ar leukocyte~ i~
isolated and the leukocytes dispersed in a ~ingle cell : su3pension. The ~ampl~ can be obtained rom any ~ite ; which contains peripheral mononuclear cell~ and must ; contain a quantity of mononuclear cells ~uch that after culturing there is an adequate number of cell~ to be analyz~d. D~nsity gradient centriugation techniques, e.g. ~coll-hypaque separation, can be utilized to inc~ea~e the p~rcentage of mononuclear cell~. Thi~
technique is ~specially important for ~amples with di~proportionate numbers of granulocytes, e.g. peripheral ~ 25 blood from subjects having a~ acute bacterial inf~c~ion or :~ from subjec~s having a disease which suppres~es the number o~ mononuclear cells.
: The sample of isolated cells is cultured with a :~ standard stimulus for a period of time suficient to allow a measurable amount of activation antigen expression~ as '' '' ' ' , influenced by mononu~lear cell subclass interaction, to ~: develop. In this particular embodiment, the isolated cell~ at a concentration of 1 million cells per milliliter are cultured with P~A at a concentration of 37.5 ~:; 5 micro~ram~ per milliliter, serving as a standard stimulu~, for 18 hours. RPMI 1640 with fetal calf serum and glutamine serve as a culture media. The culture time i determined for each particular standard stimulus using samples from normal subjects, culturing portions of each sample for different length~ of time and determining the amount of activation antigen expre~sion for each culture :: time. A measurable amount of IL-2 receptor expre~ion i8 ; observed about 18 hour~ after initiating the cul~ur~ with P~A. The 18 hour culture period facilitates the u~e of this invention as a clinical a~ay.
After culturing, the cells are wa~hed to remove the standard ~timulus and then aliquot~ of cells ar~
~imultaneou~ly incubated with two monoclonal antibodiesy each directly con~ugated to di~erent ~luo~ometrically distingui~hable fluorophores~ The fir~t fluorophore-conjug~ted monoclonal antibody binds to a selec~
mononuclear c~ll subcla3s antigenic det~rminant and the ~econd fluorometric~lly distingui~hable fluorophore-conjugated monoclonal antibody binds to a ~elect cell sur~ace activation antigen. After incubation, the cells are washed~to remove exce~s an~ibody andf in this particular embodiment, fixed with 1~ paraformaldehyde ~o halt any further cellular activity.

It should be no~ed that a portion of the isolated cells can be obtained prior to culturing.
Aliquo~s of these cells can be incubated with monoclonal antibodies and prepared as described above. These S aliquots of cell~ can then be used to measure data parameters which might be eEfected by culturin~ with a ; standard timulus, e.g. the degree of cellular activation prior to culturing. Culturing with a standard stimulus can also ef~ect changes in cell morphology and antigenic determinant modulation.
In a preferred embodiment, the first fluorophore which i~ conjugated to a monoclonal antibody is FITC and the second fluorometrically distlngui~hable fluorophore i8 ` PE. Data concerning the amount o~ gr~en and red fluorescence emitted by a particular cell as it pa~se~ th~
ligh~ beam allows for a determination of the amoun~ of ~pecific ~ubclass a~tigenic determinant~ and activation . antigens ~xpres~ed on the cell. When examining ~luore3cence data from cell~ with approximately the ~am~
:~ 20 sur~ace area (e.g. tho~ cells within an area of intere~t), the amount of antigenic determinant or activation antigen expres~ed can be referred to a~ a density.
In ~hi~ particular embodiment, direct fluorophore-conjugated msnoclonal antibodie~ are u~ed to ~: identify and enumerate cell ~urface anti~e~ lncluding : anti~Leu-3a-~ITC, anti-Leu-2a-FTTC and anti-IL2~-PE
: (Beckton Dickinson, Mountain View, C~)O Examples of : -32-alterna~ive fluorophore;conjugated monoclonal antibodies include, but are not limited to: anti-Leu-4-FITCr an~i-Leu-12-FITC, anti-Leu-ll-FITC and anti-OKT9-PE.
In this particular embodiment, an EPICS V flow cytofluorometer (Coulter Corporation, Hialeahi FL) i3 used ~o collect four parame~er data consisting of forward light catter~ orthogonal light scatter, green fluore~c~nce (515-535 nm~ and red fluorescence (~590 nm). An Inovax coh~rent argon laæer emi~ting 400 milliwa~ts of powPr at a wave l~ngth of 488 is was used as an excitation æource. A
thre~hold trigyer for forward light scatter is used to exclude r~d blood cell~ and debris. Th~ gain ~or the forward light 3catter detector amplifier i~ set at 5.
neu~ral densi~y filter is placed b~fore the forward light catter det~ctor.
: A coll~ction len~, set orthogonally to the inter~ection of the la~er beam and fluid ~tream, passes light to a serie~ of filter~ and beam ~plitt~r~O The following ~ilter. and beam splitter~ are u ~d: 488 dichroic (long pa~s) to r~flect light to the orthogo~al light acatter detect~r, a 515 .inter~erenc~ long pa~, a 560 dichroi~ ~short pass~ to re1ect light to the r~d Eluorescence detector, 590 long pa~ for red fluore~c~ce and 525 band pa~ ~or green ~luorescence. The PMT used to d~tec~,or~hogonal light ~catter i~ ~et at an applied high voltage of 330. The PMTs used to detect green and red ;; fluorescence are both se~ at an applied high voltage bf 650. The signals, from the orthogonal light scatter detector and from the green and red ~luo~escence detec~ors, are applied to integrators and ~hen logarithmic ;

D~r~

., ;: amplifiers. Prior to logarithmic amplification, a compensation n~twork to correct fQr overlap of the emission spectra of the fluorophores is used to subtract ~:: 30~ of the ~reen signal from the red siynal and 20% of the xed signal from the green signal ~or each cell.
Subtraction values are establi~hed using aliquots of stimulated mononuclear cells incubated with single fluorescent probes, e.g. anti-Leu-3-FI~C and anti-IL2R-PE.
It should be noted that the filters and setting~ ted above are exemplary and are not intended to limit the scope of the invention. However, when comparin~ ~amples~
the same particular embodiment of the method should be us~d for each sample.
The ~igures 1--11 and data presented for illustrative purposes in this detailed descrip~ion constitute an example of the assessment o the ~`: immunoregulatory ~tatus of the mononuclear leukocyte immune ~ystem of an organ transplant pati~nt. A sample was obtained as de~cribed above. Figures 1 and 2 are ~rom an aliquot of cells obtained prior to culturing with a standard stimulus. Figur~s 3-11 are from aliquQts processed after culturing with PHA for 1~ hours. The analy~i~ of these hi~tograms was performed using the MDADS
software packa~e (Coulter Corpora~ion, ~ialeah, FL~.
~ In ~IG~ 1~ we can see a histogram wherein forward and ort:hogonal light scatter is employed ~o differentiate lymphocytes, monocytes and residual ~ranulocyte~ in an aliquot from a sample of substantially mononuclear cells obtained by ficoll hypaque isolation of peripheral blood from a liver transplant patient~ These ' ' ~

~2~ o~

cells hav~ not be~n cultured with a standard stimulus~
Using the two light scatter paramet~rs the major cellular components of the immune system are differentiated.
Orthogonal light cattcr is proportional to the : 5 intracellular struc~ure, while forward light scatter is proportional to the size or diameter of the c211. The light ~catter "areas of interest" demarcated by the solid lines forming geometric ~hapes are indica~ive of the different leukocyte classes. The granulocyte class is subs~antially identified by Box 2. The monocyte class is substantiall~ identified by Box 3. The lympho~yt~ class : is substantially identified by Box 4, An aliquot of cells . obtained prior to culturing can be used to determine the quantity of ceIls in particular classes of leukocytes present in the sample of i~olated cells by integrating for the number of cells in each area of interest.
~; An ali~uot o cells obtain~d prior to culturing with a standard stimulus can be used to adjust a pr~et light scatter area of inte~est for variation~ in alignment of the ~low cyto~luorometer. The pre~et llght scatter area o~ intere~t is demarcat~d using ~orward and orthogonal light scatt~r data collected from aliquots o~
cell~ from normal individuals. Due to change~ in c~ll : morphology (e.g. cell death or the development of lymphobla ts) with increasing time in cultur~ which make the distinction between cell classes less clear on the histogram, ~he preset light scatter ar~a of in~erest is adjusted u~ing an aliquot of cells obtained prior to ~: : culturing. This adjusted area of interest is then used to 30 analyze all aliquots of the sample, including those -35~

obtained aft~r culturing. In cases where the entire ~ sample mu~t be put into culture due to low cell number, ; the pre~et li~ht ~catter areas of inter~t i5 used ~o analyze the aliquotæ of cells.
FIG. 2 shows the same histogram as in FIG. 1 except that only ~he area of interest indicative of lymphocytes is identified, i.e. Box 7 (the same as Box 4 ~ in ~IG. l). The x and y coordinates of the point~ used to ;~ generate the g~ometric shape are list~d to the left of the - lO histogram. These are the coordinates of the pre~et light ~: scatter area of interest substantially identifying lymphocyte~ used in this particular embodiment. There was no adjustment of the pre~et llght scatter area of interest for this sample. Of the 15~784 to~al cell~ represented on the histogxam, 10,132 cell~ or 64.19 percent are within ~; the area o~ inter~t which ~ubstantially ide~tiEies lymphocytes.
By mea~uring the two light scatter and two fluorescent parameters, th~ ~low cyto~luorom~ter gener~tes four parameter data on individual cells which can be analyzed to determine activation antigen expre~sion on cells of a s~lect mononuclea~ c~ll 3ubclass. When this four parameter data for individual eells is g~ouped for : all of.the ~ell~ of the ali~uot for which data was generated, it i~ termed cumulative four parameter data.
~;~ Analysis of the cumulative four parameter data for an aliquot of mononu~lear cells identifies cells of a select mononuclear cell class(es) using li~ht scatter area~s) of ~ interest, e.g. lymphocytes and/or lymphoblasts. Cells of -: 30 the particular clas~(es) are then analyzed to determine ~2~

the minimal density of antigenic determinant expre~ion on a cell necessary tQ qualify the cell as a member of a selec~ mononu~lear cell subclass, the quantity o cells having greater than the minimal density of antigenic : 5 determinant, and the quantity of activated cells in said s~lect mononuclear cell subcla~s having greater than a preset minimal density of activation an~igen expre~sion necessary to qualify the cell as being activated.
In a similar manner, analy~i~ o cummulative four parameter data can be performed for aliquots of cells ob~ained prior to culturing with a ~tandard simulus. It is usually as~umed that there i~ little ~ignificant activation antigen expression prior to culturing with a standard stimulus, however in patients with certain dlsease~ or receiving immunopotentiating medication, there could be an appr~ciable amount of activation antigen expre~ion. In thes case~, analysis o the degree of cellular activat.ion in particular ~ubcl~sses should be : perormed prior to and after culturing with a ~tandard stimulu~ such that the amount o change in ~ellular activation can be det~rmined. Al~o, an analy~l~ to determine the guantity of cells in particular mon~nuclear cell subcIass~s can ~e perfor~ed prior to culturing with a standard stimulus which can modulate the pa~ticular antig~hic determinants.
Figures 3~9 illu~trate an analysis of cumulative four parameter daka for cells from an aliquot vf cells cultured with PHA for 18 hours and then incubated with anti-Leu-3a-FITC and an~i-IL2R-PE~ In a preferred embodiment, green and red fluorescence data i5 analyzed , ~

~: for apprsximately 10,000 cells whose light ~catter data - placed them in the "lymphocyte" area of interestO This ~ increases the ~tatistical significance of the analysis of ;~ this data. FIG. 3 shows a two parameter hi~togram of unanalyzed forward and orthogonal light scatter parameters for the 22,199 cells on which data was collected.
FIG. 4 shows the same histogram as i~ FIG. 3 with the area of inter~st which substantially ~dentifie~
l:ymphocytes demarcated, i~e. Box 19. The x and y coordinates of the points used to generate the geometric shape are listed to the left of the histogram. These coordinate are the same as those used to generate the shape on a similar histogram of cells prior to culturiny (see FIG. 2). For every aliquot of cells from a particular sample, bQth before and aft~r ~tlmulation, the coordinates used to g~nerate an area of int~rest on the forward and orthogonal light scatter hi~togram are the same such that the area o~ interest demarcate~ cell~ of ~imilar morphology for each aliquot~
By defining a minimal ~and maximal) density o~
speci~ic antigenic d~terminant for inclusion in a certain subcla~s, cells c~n be assigned to the ~ubcla~s by measuring the amount o~ fluorescence associated with the fluoro~hore-conjugated monoclan~l antikody which identi~ie~ the antigenic determinant on the cell's surface. The assigned cells are reerred to as being : ~ antigenic dete~minant positive. ~150, a minimal de~sity can be deEined for activation antlgen expression to determine the pres2nce of such antigens. Certain ranges of density above this minimal are measured for certain ?trr~/;

activatlon antigens~ e.g. IL2R, and can serve as an indirect de~ermination of ~pecific lympho-monokine ~ production, e.g. IL-2.
:~ The density of a certain specific antigenic determinant can vary somewhat between individual~ or with antigen modulation, e.g. during activation. To determine the minimal density of a ~pecific antigenic determinant for a particular aliquot of cells which have been incubated with ~irst and ~econd ~luorophore-conjugated ~; 10 monoclonal antibodies, a plot of cell number versus amount of first fluorophore fluorescence i5 made. In this particular embodiment, the first fluorophore i~ FITC and ~ it i8 conjugated to monoclonal antibodies identiying : anti~enic determinant9. The second ~luorophore i~ PE and ~ 15 is conjugated to monoclonal antibodies identifyin~
:~ activation antig~n~. ~sing only cells which have leR~
than a certain minimal amount of red fluor~ n~e will ~ exclude many o~ the nonspecific binding cell~ a~ they will ; hav~ bound both the green and red fluorescent antibodies~
From the plot o~ cell number versu~ th~ amount o~ green fluore~cen~e, an estimate o the tangent to the positive slope of the curve can be made. The curve represent~ an approximate gaussian distribution due to the lognormal ~ di~tribution of anti~enic determinants. The x-intercept :~. 25 generated by such a tange~t will defin~ ~ minimal f~uore~cence intensity which correlate~ with a minimal spe~ific antigenic determinant density. (A similar procedure can be used to determine the maximal antigenic determinan~ density from the negative ~lope.) In this :; 30 particular embodimen~/ the maximal den5ity is set as the highest density which can be measured. Using the~e density boundaries, the two parameter histogram of green and red fluorescence can be integrated between these boundaries to enumerate the number of cell~ which are positive for the antigenic determinant and can be assigned to a particular subclass. It ~hould be noted that alternative methods can be used to determine these minimal and maximal densities such that a similar number of cells will be assigned to the subclass.
A minimal density is defined to determine the presence of activation antigen This minimal density will vary with different gain and high voltage settings for certain components of the flow cytofluorometer and the particular light ~ource, alignment, filter~ and lS fluorescent detection electronics. Thu , lt c~n be ~en that this minimal density o~ activation antigPn expre~;on must be de~ined a~ter determinin~ standard settings and will be indicatlve of a minimal density mea~urable by a particular flow cytofluorometer using these sett.ing~.
An iterative analysis o~ sample~ from normal individuals, both prior to and after culturing with a standard stimulu~ for a certain length of time, mu3t be performed to determine the minimal density o~ activation antige~ measurable. This analysis use~ the two parameter fluorescence data ~rom aliquots of cells incubated with : fluorometrically distinguishable fluorophore-conjugated monoclonal antibodies either to both the antigenic determinant and the activation antigen, to both the antigenic determinant and a control te.~. non-human) antigen, or to only the antigenic determinant. From two ' : parameter hi tograms a minimal fluorescent in~ensity indicative of activation antigen expression can be determined above which there is more speci~ic (versus nonspecific) binding and the overlap from the emission -: 5 spectra the fluorophore indicative of the antigenic determinant of is not significant ~e.g. less than about 2 percent).
~ hus, each aliquot is first analyzed ~o determine the minimal (and maximal) density of a specific antigenic determinant for inclusion in the subclass being examined and the ~ubclass i5 enumerated from the two :~ parameter fluorescence data. Then by further analyzing the two parameter fluorescence data using these densities and the preset minimal activation anti~Qn density, an enumeration o the number of oells which belong to the subclass and have expressed activation anti~en is made.
In a similar way, an enumeration i~ made of the number o~
cell~ which belong to the subclass and have expressed :~ acklvation antigens within certain pre~et speci~ied den~ity r~nge3. Cum~ulativ~ four parameter data ~rom an aliquot inaubated with both ~irst fluorophore- and ~econd fluorophore-conjugated control monoclonal antibodi2s is analyzed u~ing these ~ame densities to determinP th~
approximate number of fals~ positives due to nonspecific binding. If the amount of nonspeciflc binding is :~ significant, the result~ obtained ~rom aliquots incubated with the monoclonal antibodie~ to antigenic determinants ~; and activation antigens can be corrected.
~:

2~

~41-It should be noted that cells with two ~pecific antigenic determinants of known den~ities can be id~ntifi~d with two different monoclonal antibodies conjugated to the ~ame first fluorophore such that the cumulative fluorescence emitted is equivalent to the sum of their respective densities. This ~echnique allows further distinction of suhcla~ses, e.g~ cells having both Leu-3a and Leu-8 antigenic determinant~. As above, ~he second fluorophore-conjugated monoclonal antibody identifies a certain activation antigen which might be expressed o~er a range of densities. Alternatively, three fluorometrically distinguishable fluorophore-conju~ated monoclonal antibodies can be u~ed to identiXy two apeciic antigenic determinants and an ac~ivation antigen~
In this particular embodiment, green and red fluorescenc2 data is analyzed only for cells meeting the ~orward an~ ortho~onal light scatter criteri~ a~ deined by the arsa of interest ~ubstantially identi~ying ~he lymphocytes. FIG. 5 shows a two parameter hi~togram of una~alyzed green and red fluorescence data parameters of the leukocytes identl~ied to be lymphocyte~ in FIG. 4.
The green and red fluore~cence lntensitie~ relate to the number of excited fluorophore~ bound to the cell by ~he monoclonal antibodies anti-Leu-3a-FITC and anti-IL2R-PE, respec,tively.
In this particular embodiment~ a plot of cell number versus the amount of green fluorescence i5 constructed using only those cPlls in the zero and one red fluorescence channels. An estimate of the tangent ~o the ~- 30 positive 510p2 of the curve is made. The x-intercept i6~.~

generated by this tangent defines the minimal specificantigenic determinant densityO FIG. 6 show~ a one parameter hi togram of the green fluore~cence data ; parameter of the lymphocytes for which the amount of red fluore~cence measured placed them in the 0 or 1 channel on the LRF axis. An approximate bell-shaped curve is generated. An approximation of the tangent to the positive slope of the curve is used to generate a line, 21. It ~hould be noted that the curve can be processed using computerized smoothing techniques to facilitate this approximation. The approximate x-intercept of the line, channel 20, is used as the channel number at which to draw a cur~or line on the two parameter histogram in ~IG. 7.
This is con~idered the minimal den~ity o antigenic determin2nt expre~3ion which a cell must have to qualify as a member of the sel~ct mononuclear ~ubclass, which the antigenia dekerminant defines.
A cur~or i~ set on the green and red fluore~cence data histogram using the x~intercept. FIG~ 7 ~hows the ~ame two parameter hi~togram a~ in PIG. 5 with a cur~or line 22, set at LGF chann~l 20 a~ determined in FIG. 6. A5 ~hown in FIGo 7, the area of intere~t used to determine the percenta~e of lymphocytes which can be as~igned to the subcla~ identified by antigenic determinant Leu-3a, i.e. Leu-3a positive cells (hereinafter Leu-3a+),:is between LGF c~annels 20 and 63.
The percentage of lymphocytes as identified in FIG~ 7 which are Leu 3a+ is 21.81.

66~

-~3-- In this particular embodiment, a red fluorescence cursor i set at channel 10 on the green and red fluorescence data histogram and the number of ~otal cells, i.e. lymphocyt~s, which are activated and are 5 as.~igned to the subcl~ss identified by the antigenic determinant is determined. A LRF channel a~ 10 was chosen to represent the minimal amount of IL-2 receptor which a : cell must expre~s to be onsidered activated, after an iterative analysis as detailed above. FIG. 8 shows the same two parameter histogram as in FIG. 7 with a second cursor line, 25, set at LRF channel 10. A~ shown in FI~.
8, the area of interest used to determine the percentage of lymphocytes which are ~eu~3a~ and expres3 greater than the minimal amount of IL-2 receptor (herein~fter IL2R~
between LGF ~hannelq 20 and 63, and ~RF channels 10 and 63. The percentage o~ lymphocyte~ which are Leu-3a~ and IL2R~ (or L~u-3a~IL2R+) is 20.670 In this particular embodiment r a r~d fluorescence cur~or is 3et at channel 25 on the green and red fluorescence data h.istogram to di~tingui.3h di~ferent density ranges of IL-2 receptor expres~ion o~ cell~ which are member~ o~ a select subGlaa~. The quantity of lymphocytes which are members of a select ~ubcla~s and have a,high de~sity of IL 2 receptor expression i.R
de~ermined. The channel number 25 was determined usin~ an iterative analysi of sample~ from normal individuals cultured for 48 hour~ to examine for the average den~ity range present on such cells. A LR~ channel of 25 was cho~en to repre~ent the lowe~t density of IL-2 receptors which a cell must express to be considered to have a high :

density of IL2R on it~ cell surface. FIG. 9 shows the same two parameter histogram as in FIG. 7 with a third cursor line, ~8, set at LRF channel 25. As shown in Figure 9, the area of interest used to determine th~
percentage of lymphocytes which are Leu-3a+ and express a : high density of IL-2 receptor is between LGF channelR 20 and 63 and LRF channels 25 and 630 The percentage of lymphocytes which are Leu-3a+ and express a h;gh density of IL 2 receptor ls 8.16.
A ~imilar analysis of cumulative four parameter data as that shown in figures 3-9 can be perPormed for each aliquot from the sample of peripheral mononuclear cells.
In this particul~r embodiment, cells with a high density o~ ~eu-~a antigenic determinants are con~idered to ;~ be sf the suppressor cytotoxic T-c~ll subclass. FI~. 10 show~ a two parameter f}uores~ence data hi~togram similar to that in FIG. ~ for an aliquot of cell~ rcm the same sample and which wa~ incubated with anti-Leu2a-~ITC and anti-I~2R-PE~ The green fluorescence cursor 30 iR ~t ~t channel 37 and the red fluorescence cur~or 31 i~ ~et at channel 10 on th~ gresn and red fluorescence data hi~togram. The area of interest u~ed to determine the percentage of lymphocytes which are ~eu~2a+ and IL2R+ i ~S be ween LGF channel~ 37 and 63 and LRF channels 10 and 63.
The percentage of lymphocytes which are Leu-2a~ and IL2R+
is 7.96.
he occurrence of nonspecific binding of the monoclonal antibodies, which oft~n increases with the time spent in culture, vaFies for the different leukocyte .
.

'' ' , ' , , .

:

-~5-classes. Granulocytes and monocytes have relatively higher densities of nonspecific binding sites on their cell surface~ than do lymphocytes. This nonspecific binding makes accurate cytofluorometric measurement of activated cells more difficult as the av~rag~ density of nonspecific binding sites nears th~t of the antigenic determinants and activation antigens. An accurate determination, however, can be made using forward and orthogonal light scatter parameters to delineate the leukocyte classes and excluding the classes with high densi~ies of nonspecific binding sites when analyzing fluorescence data for lymphocytes which have low densities of ~uch ~ites. It ~hould be noted that an unconjugat~d monoclonal antibody with high affinity for a nonspecific bind~ng site, ~.g. anti Leu-ll Eor the granulocyte and natural killer c~ll E'c receptor or a monoclonal antibody to the monocyte Fc receptor, can be u~ed to block the site's low affinity non-speciic binding with other monoclonal antibodies.
In a similar manner to the analy es de~cribed above, fluorophor~-conjugated control monoclonal antibodie~ directed against antigens not found in ~he human immune system can be u~ed to determine the number of ;~ cells with a significantly high quantity nonspecific ~:~ 25 binding site densitie3 su~h that these cells are in the areas of interest. FIG. 11 shows a two parameter histogram of unanalyzed green and red fluorescence data parameter data of the leukocytes identiEied to be ~ lymphocytes similar to FI~ 4. The green and red ;~ 30 fluorescence intensities relate to the number of excit0d , fluorophores bound to the cell by the control monoclonal antibodies, mouse-IgG1-FITG and mouse-IgG1-PE, respectively (Becton Dickinson, Mountain View, CA). The same areas of interest as used in figures 7-10 were applied to this histogram thereby determining an approximate number of false positives in each area of interest which is due to nonspecific binding. This allows for correction of the percentages determined for each area of interest in figure 7-10.
From the analysis of cumulative four parameter data for each aliquot, the degree of cellular activation of each mononuclear cell subclass can be determined. This can be used to assess the immunoregulatory status of the mononuclear leukocyte immune system of the sample of peripheral mononuclear cells and can be correlated with the in vivo immunoregulatory status of the patient as determined clinically. In this illustration of a preferred embodiment, the degree of cellular activation for each subclass is determined using the percentage of a select subclass which are activated and ratios of the percentage of activation for the different subclasses.
This is utilized to clinically assess the immunoregulatory status of the mononuclear leukocyte immune system of liver transplant patients. In this example, the percentage of cells of a select mononuclear cell subclass which are activated, i.e. express greater than the preset minimal IL-2 receptor density, are termed "Activated Leu-3a+", etc.

6~'J

Peripheral blood samples were obtained from liver transplant recipients sequentially over a period of four months after receiving the transplantO Ten normal individuals ~erved as controls. Mononuclear leukocytes from each sample were isolated, cultured~ incubated with monoclonal antibodies and four parameter data coll~cted and analyzed a~ described above. Certain results for the patient sample illustrated in figures 1-11 are found in Table I.

lQ TABLE I
Rat10 Activa-ted Leu-3af Actlvated Activated :Activated 15 Li~er Transplant Recip1ent A 73 26 2.8 From Tabl~ I it i~ seen that 73 percent of the patient'~ helper/inducer lymphocyte~ became activa~ed aB
measured by IL-2 receptvr expre~sion after culturing with PHA for lB hour~. Similarly, 26 percent of the p~tient's cytotoxic/suppre~sor T lymphocytes hecame activated. The ratio ~ the percent activation of the two subsets was 2.~.
: The results of the present assay were correlated with liver biop~y patholoyic determinations of rejection obtained on the same day as the samples. Th2 morphologic criteria for a diagnosis of "no rejection" included normal or mild nonspecific portal inflammation consisting mainly .

-4~-of lymphocytes. The morphologic criteria for a diagnosis : of "rejection" included inflammatory infiltrate of triads with lymphocytes and plasma cells resulting in ~iecemeal necrosis, disorganiza~ion and degenerative changes of bile duct epithelium, and phlebitis of cen~ral veins. A
diagnosis of "suspicious" was given to those samples with partial criteria for a diagnosi~ of rejection.
The results of th~ generated and analyzed ~our parameter data from aliqouts of samples from liver transplant recipi2nts are summarized in Table II. Samples were included in the "No Rejection" category on the table if the corresponding biopsy had a diagno~is of "no rejection" and the s~mple was not obtained within 7 days : of transplant surgery or within lO days of a "suspicious"
:. 15 or "rejection" biopsy diagnosis. The samples in the "Rejection" category were obtained from patient~ who were on s~able do~es of immunosuppressive medications (glucocorticoid~ and cyclosporin) and had a corresponding biopsy diagnosis of "rejection".

~'~

, .. . . . . .

~49--TABLE I I

Ratio Act1va-ted Leu-3a+
Activated Activated :A~tivated S l~3a+ Le~-2a~ Lel-2a+

Normals (n=10) 75 ~ 6 ~ 4 4 to 14 L1ver Transplant Recipie~ts No Rejection (n=13) 61 ~ 18 9 + B 2.8 to 15 Rejection (n=6) 80 + 9 37 ~ 13 1.5 to 3.0 : 10 ~ mean + standard deviation.
:
It can be seen from the ~ummarized results in Table II that liver rejection correlates with a higher percent of activated Leu-2a~ lymphocyt~s, i.e. the ; altered immunoregulatory status can be asses~ed from the degree of Leu-2a-~ cellular activation. Therefore, u~ing the method~ d~cribed herein the clinician can assess the immuno~egulatory ~tatu~ of the mononuclear leukocyte immune system to facilitate the diagno~i~ of transplanted oryan re~ection.

Example_2 : This example shows an analysi~ of the immunoregulatory sta~us of the mononuclear leukocyte immune system of patients receiving immunosuppression ~: therapy. Peripheral blood samples were obtained from ten normal individuals and six liver transplant recipie~ts.
~:The transplant recipients are divided into 2 groups. The first group consists of 4 patients who did not have a rejection episode in their four to ~ight week post-transplant hospital course. The second group consis~s o two patients with two sequential samples both with corresponding biopsy dianosis of "rejection".
Peripheral blood mononuclear leukocytes from each sample were isolated by density gradient centrifugation, cultured with P~ for 18 hours, i~cubated with monoclonal antibodies and four parameter data ~;collected and analyzed as described in "Detailed Description". Unless otherwise not~d, all sampl~s were obtained from the patients about one hour prior to receiving their immunosuppressive medication. Biopses were obtained and pathologic determination~ done as described above.
Each normal had one sample drawn while each o~
the irst group of transplant recipient~ had Eour to eight samples drawn on differ~nt date~ during their ~our to eight week post tran~plant ho~pital cour~e. During their hospital course, the p~tients were treated with cyclosporin and corticosteroids for imm~nosuppressive therapy. Cyclo~porin blood level~ wer~ monitored u~ing ~25 the HPLC method~ G.L. Lensm&yer and B.L. Fields, :~"Improved Liquid-Chromatographic Det~rmination of Cyclosporin, with Concomitant Detection of a Cell-Bound Metaboli~e," C_inical Chemistr~, (31) 196-201 ~1985).
~' ~',' .
During their hospital course there were certain changes in the cyclosporin dose, cyclosporin level and/or corticosteroid dose.
The pertinent clinical data and results are summarized in Table III. The following should be noted:
all cyclosporin doses were converted to the equivalent intravenous dose using a conversion factor of 1/3 for oral doses ~Physician's Desk Reference, 1986) and ~11 glucocorticoid doses were converted ~o the predisone equivalent (p. 365, Manual of ~edical Th~ra eutic~, 1983). A11 samples are grouped by patient to facilitate analysis of changes in immunosuppressive th~rapy~ Also, all samples are }isted sequentially startin~
:~ approximately one week after receiving the liver ~: 15 transplant. Samples were obtained ev~ry three to seven days. For those patients tested, the values ~or the analysis of samples obtained prior to transplant ; approximated the normal values~
~' .:

t3~

Cyclo- Cyclo- Pred % of Activated sporin sporin n;sone ActiYated* Leu-3a~ expressing a Pat~entDose ~lQQd_L~el Dose Leu-3a+ h;gh densi~ of I~2 Normal 0 0 0 75 + 6 40 _ 9 (n-10) 6(n~4~lZ0 228 + 42 25 7 14 ZO(n=3) 28 ~ 4 17 + 1 - H(n-8) 80 122 ~ 31 38 15 13 z5(n-5) 35 + 8 31 + 6 ZO(n=2) 74 + 5 31 + 6 I(n~4)180(n~2)~176 + 40 20 62 + 4 1~
` 170(n~2) 408 + 106 13 ~ 4 14 ~ 7 ~; J(n=8)200 Z54 50 45 31 ~`` 200 101 25 7~ 38 ~"' 125 ~00 19 Ul 36 ~' 125 90 19 73 3~
`~ 75 43 13 73 66 0 0 ~ 63 Act~v~ted Leu-3a~ ~ the percentage of Leu-3a~ which expr~ss gre~ter than the minlmal IL-2 receptor denslty.
~ ~e~n ~ standard dev1ation ~ Thls pat1~nt had unexpectedly high oral absorpt~on incre~s~ng hls blood 1evel.
Cyclosporin dose ~ mg every 12 hours, level 1s ng/ml ~ Prednisone dose ~ mg every 24 hours.
:: ~ From this study it can be seen that the m~thod 30 described herein detects changes in the immunoregulatory ~tatus due to immunosuppressive therapy. From the sumrnarized results in Table III, it can be seen that changes in immuno~uppressive therapy ha~7e a neyative correlation with changes in the percent of activated Leu-. .

: ~, 3a+ lymphocytes and of activated Leu-3a~ expressing a high density of Ih-Z receptor, e.~. increase~ in immunosuppre~sive therapy yields a decrea~e in activated L~u-3a+. In patients G and ~, the cyclosporin dose and level~ were approximately onst~nt while the glucocorticoid (predisone) dose was decreas@d. It is ~een that the percent of activated Leu-3a+ lymphocytes increase~ with decreasing doses of glucocorticoids in the~e patien~s~ In patient I:there was unexpectedly high oral absorption of cyclo~porin when the patient was converted from intravenous to oral doses. ~he patientqs cyclospvrin level increased while the glucocorticoid dose remained con~tant~ It is ~;~ seen that the percent of activated Leu 3a~ lymphocytes decreased with increa~ed blood levels of cyclosporin in lS this patient. In patient J, eight ~ampleæ are listed sequentially ~howing d~crea~in~ cyclosporin dose, cyclosporin level ~nd glucocorticoid do8e. It ~n be seen that the percerlt o~ activated Leu-3a~ lymphocyte~ increase~
with the decreasing level of immuno~uppre~ive therapy in thi~ patient. It can al~o be seen that a~ the cyclo~porin level and glucocorticoid dose near zero that th~ percent of activated Leu-3a+ lymphocytes expresslng a high density of IL-2 receptore increas~s/ possibly related to ~he relPase of the suppress:on o~ IL-2 production by the immunosuppressive medication~. The increased level of IL-2 production would thereby unr~gulate the IL-2 receptor.

The results from the sample~ drawn from the second group of transplant recipients show the acute ~ effects of immunosuppressive therapy even during rejection : episodes~ The pertinent clinical data and xesults are summarized in Table IV.

TABLE TV
~;~ Cyclo- Cyclo- Pred X of Activated sporin sporin nisoneActivated* Leu-3a~ expressing a Pati~n~ Dose ~lQod Level ~Q~ Leu-3a~ high densitY of ILZR
~ 10 A~* 68 200 15 73 39 : A 68 N.D. 15 66 10 B~ 150 217 25 91 69 B 150 325 1,250 82 38 See Table I~I ~or descrlpt~on.
- ~ 15 ~* Th~s sample was drawn about Z hours ~ft~r th~ patient h~d received h1s ~mmunosuppressive med~cDt10n.
~* Th~s sample was dr~wn about 18 hours after the patlent h~d recelved a very large dose of :~ cort kosteroids.
N.D. ~ not done Cyclospor1n dose ~ mg every 12 hours, level is ng/ml.
Pr~dn1sone dose ~ mg every 24 hours.

The methods described her~in detect changes in ~: the immunoregulatory 5tatu5 due to the acute effects of immunosuppressive therapy even during r jection episodes.
The acute effect~ of immunosuppressive therapy rorrelate ~ with a decrease in the percent of activated Leu-3a+
:~ ~ lymphocytes expressing a high densi~y o IL-2 recep~or.

'~

~2~

FIGS. 12 and 12A show histoqrams similar to FIG. 3/ generated from the samples from patient B listed in Table IV. By comparison, it is ~een that the densi~y `~ distribution of IL-2 receptor on the ~eu-3a+ subclass is different for these two samples from the same subject on different dates. FIG. 12A was generated from the ~ample drawn 18 hours after the large does of glucocorticoids.
Analysis of the four parameter data used to generate FIG.
12 revealed that 29.5S percent of the cells were Leu-3a~, 26.87 percent of the cells were Leu-3a+ and IL2R~, and ~ 18c49 percent of the cells were Leu-3a~ and expressed a :~: high density of IL2R. A similar analysis of the four parameter data used to generate FIG. 12A yielded 29.59, 22.93 and 7.~0 percent, respectively.
The result~ shown in this example illustrate ~at the m~thods described herein can be used to monitor the effects of immunosuppressive therapy on the immunoregulatory tatus of the mononuclear leukocyte immune ~ystem of pati~nts. ~y delineating a ~elect mononuclear cell ~ubclass, this assay can facilitate the target:Lng o~
immunosuppresive or i~munopotentiating therapy ~or a particular subcla35. Also, altexations of the methods described herein, ~.g. u~ing serum from a patient on immuno8uppres Lve or immunopotentiating therapy in the culture, can ~a~ilitate a refinement of the a~sessment o~
~; the effects of a patient's medication vn the immunoregulatory status and are within the scope of this invention~
: :

:~
~,'' ' ' ~ , , ' ' ' r~

.~
Example 3 This example shows an analy~is of T lymphocyte antigenic determinant expressior., IL-Z receptor expression and IL-2 receptor expression on T lymphocyte antigenic determinant positive mononuclear cells. This analysis is correlated with the stimulation index as ~easured by a 72 hour P~A culture with tritiated thymidine uptake. Peripheral blood mononuclear cells from normal donors were isolated by density gradient ~ 10 centrifugation, cultured with P~A, incubated with :~ monoclonal antibodies to select mononuclear cell subclass `i antigenic de~erminants and to I~-2 receptors and four parameter data generated and analyzed similar as described in "Detailed Description". Monoclonal antibodies used included FITC-conjugated antibody for the antigenic determinants Leu-4~ Leu-3a or Leu-2a, and PE-conjugat~d monoclonal antibody or IL-2 receptorO Four parameter data wa~ collected on cell~ using an EPICS~-V
~:~ flow cyto~luorometer (Coulter Corporation, EIialeah, FL) con~igured a~ described in "Detail~d Description" with the following modification: the argon laser was used at 500 milllwatts. Forward and orthogonal light ~catter parameters were analyzed to determine if the cell was ~ within, an area of interest which substantially încluded :~ 25 lymphocytes, lymphoblasts and monocytes, Green and red fluorescence data analysi~ was performed for such cells.
The percentage of antigenic ~eterminant and/or IL-2 receptor positive mononuclear cells, i~e., Leu-4+, Leu-3a~, Leu-2a~, IL2R+, leu-4+IL2R~, Leu-3a~IL2R+ arld Leu-, ~ : ' ~ -57-;`~ 2a~IL2R~, was de~ermined as per the methods described ~: herein. The results were not corrected for non-specific ~; bindingq : First, mononuclear cell subclass antigenic determinant and activation antigen analysis of PHA
stimulated mononuclear cells was conducted using aliquot~
incubated with only one monoclonal antibodyO The results ; are summarized in Table V.

TABLE V
Time in Culture with P~A ~in hours) . Leu 4~G8 ~ 2.7* 79 + 2.4 77 + 1.981 + 2~2 Leu 3a~47 ~ 2~9 $4 + 1.7 54 + 1.353 ~ 2.8 Leu 2a~13 + 0.7 1~ ~ 1.0 18 ~ 0.719 + 2.0 lS IL2R~6 + 1~2 61 ~ 1.9 71 ~ 1.980 ~ 1.5 * Expres~ed as p~rcentage positive + standard error.
N - 9.
From the above study of cells stimulated for various time~, as lon~ as 48 hour~, it was ~ound that ~ : 20 there ,wa~ significant interleukin-2 receptor expression :~ a~ early as 18 hours after initiating activa~ion. At : about 1~ hours it was observed that nearly 60% of the mononuclear cells in the area of interest expressed XL-2 ~ receptor~.

,' ' ' ' . : .

~,, 6~

:.

.~
Analysis of the PH~ stimulated mononuclear cells was conducted using aliquots incubated with a FITC-conjugated monoclonal antibody to detect antigenic determinants and anti-IL2R PE. The results are summarized in Table VI.

~ABLE VI

Time in Culture with PHA (in hours)*
Q 1~ 24 4 ~ Acti-.~ lO vated **
Leu-4~ 1 + 0.3 49 t 6~1 62 ~ 3.7 72 ~ 5.5 ti-vated Leu-3a~ 3 ~ 1~7 49 * ~.9 62 ~ 8.5 70 + 9.5 15 Acti-~; vated Leu-2a~ 2 + 0.8 25 t 6.4 32 ~ 4.5 55 * 8.0 * At 72 hours post stimulation the stimulation index was determined u~ing tritiated thymidine uptake (cpm stimulated/cpm un~timulated cells). The result wa~:
367 + 88. ~For comparison, the re~lllt at 18 hours was 2.75 ~ 0.25).
** Percentage of antigenic determinant positive cells whi'ch expre~s greater than the preset minimal IL-2 .~ 25 receptor density.
~: These data support previou~ ~tudies that P~A
. ~
preferentially activates helper/inducer T-cells as shown in this example by mononuclear cells which are Leu-3a~0 It should be noted that the ~our parameter data used in ~, ~59-this example was analyzed using a light scatter area of ~: interest which susbstantially identified all ~ononuclear :~ cells, therefore the results for activated Leu-3a+ and activated Leu-2a~ (Tables VI, VII) are not identical to re~ults determined using a light ~catter area of interest identifying lymphocyte~. Analysis of IL-2 receptor expression on antigenic determinant positive mononuclear cell gives more information concerning the kinetics of the mononuclear cell response to mitogens than does conventional tritiated thymidine uptake.
An Activated Leu-3a~-Activated Leu-2a~ ratio greater than 1.4 correlated with a high s~imulation index (hereinafter SI) tG PHA (>400). Cell~ from one subject had a SI of 8 and a ratio of approximately 1.0 suggesting ~hat one could predict mononuclear cell proliferating :: ability a~ measurcd using ~he 5I determined at 72 hour~
by measuring IL~2 receptor expression on antigenic determinant po~itive mononuclear cells at 18 hour~ to :: compute a ratio. This relationship i~ su~nari~ed in Table VII.

.
;

,, ~?~

~ -~0-Table VII

; SI
0+ 18 24 48 72 Subject D: ~cti-vated *
Leu-4~ 1 68 75 84 535 Acti-vated Leu-3a+ 7 74 79 92 ~: Acti-~` vated eu-2a+ 1 32 32 76 ratio** 2.31 ~:.
Subject N: Acti-vated Leu-4~ 1 32 59 ND
Acti-:~ vated ~eu-3a+ 1 31 ND ~4 ; ~cti-vated Leu-Za-~ 2 30 ND 71 ratio 1~03 ~ ~ours in culture with P~A.
Percentage of antigenic determinant positive cells :which express greater than the preset minimal IL-2 receptor density~
~ ~ ** At 18 hours the ratio of Activated Leu-3a+:Activated ;~ 30 Leu-2a+ ells was determined for each sample.

, . . .

From the above, it is seen that there is a differential activation of anti~enic det~rminant positiqe mononuclear cells stimulated with PHA which can be measured by examining IL-2 receptor expression on ~uch cells. Analysis of IL-2 receptor expression on these mononuclear cells after PHA stimulation gives a better understanding of the kinetics of the cellular activation in the mononuclear leukocyte immune system.

Exam~le 4 This example shows analysis of IL-2 receptor expre~sion on the lymphocyte claQs compared to I~-2 : receptor expre~sion on T lymphocyte subclasses. ~ sample Q~ peripher~l blood mononuclear leukocytes from a normal donor and a liver transplant recipient were isolated using density gradi~nt centriEugat.ion, cultured with P~A for la hour~ and incubated with monoclonal antibodies as de~cribed in "Detailed De~cription". Ali~uots of the ~ample ~rom each individual were incubated with either anti-IL2R-PE~ anti-Leu-4-~ITC and anti-II,2R-PE, anti-Leu-3a-FITC and anti-IL2R-PE, or anti~eu-2a~FITC and anti-IL2R-PE (Becton Dickinson, Mountain Viçw, C~). Aliquots incubated wi th mouse-IgGl-PE, or mouse-IgGl-FITC and mou~e-IgGl PE 5~ecton Dickinson, Mountain View, CA) served ;:as controls to determine nonspecific binding, Four :~25 par~meter data was collected on cells from each aliquot using an EPICS-V flow cytofluorometer ~Coulter Corporation, Hialeah, FL) conflgured as described in "Detailed ~escription". Forward and orthogonal light ''' scatter parameters were analyzed to determine if the cell was in a light catter area of interest which ubstantially identified lymphocytes. Green and red fluore~cence data analysis for aliquots incubated with two monoclonal antibodie~ was conducted as described in "Detailed Description". Fluorescence data analysis for aliquots incubated with only anti-IL2R-PE was similar, ~: ex~ept that the minimal amount of I~2R expression was :~: determined using simple one paxameter (red fluorescence) :~ lO subtraction from the fluorescence generated by the aliquot incubated with only mouse-IgGl-PE.
~: The red fluorescence data relates to the number of "activated" lymphocytes. With respect to FIG. 13, this i~ a histogram wherein IL-2 receptors are mea~ured on lymphocytes identified similarly as in FXG. 2 after the sample o~ mononuclear c~lls was cultured with P~A for 18 hours. The red fluorescent inten~ity relate to the number of ~xcited fluorophores bound to the cell by ~he :~ monoclonal antibody, anti~IL2R-PE. This igure was gen0rated from an aliquot ~rom the normal ~ubjeot~
~ he resulta o~ this study are summarized in Table VIII. ,~

~.3~2 TABLE VIII

Activated** Activated Activated Subie tIL2R~* Leu-4+ _~y~a+ Leu-2a+
Normal 49 61 55 9 5 Liver Transplant Recipient A 39 74 73 26 * Percentage of lymphocytes which expres~ed greater than ~he minimal IL 2 receptor density a~ d~termined by one parameter sub~raction.
~: ** Percentage of the antigenic determinant positive lymphocytes which express greater than the preset minimal IL-2 receptor den~ity.
:~ It is seen that the percentage of lymphocytes expressing XL-2 receptor does not neces~arily cvrrespond ~ to the p~rcentage of ~elect ~ lymphocyte ubclasses : expressing IL-2 receptor. There i8 a differential : activation o T lymphocyte subclas~e~ cultured wlth P~
which cannot be determined by measuring IL-2 receptor expre~sion on cells identifi~d only as lymphocyte~.
}Iowev~r, it can be d~termined by e~amining lL~2 receptor expression on ~uch subclasse~.
.
Exam~le 5 This example shows analysi~ o activation antigen expression on particular monocluear cell subclasses after culturing with PHA for 18 hours. This ; analysis correlates with a known clinical assay, tritiated , ,,,,~ , 6~

-6~-tAymidine uptake after culturing with P~A 72 hours, ; however~ yields more complete information concerning ~heimmunoregulatory status of the mononucle~r leu~ocy~e immune systemO
Samples of peripheral blood from a normal individual and a liver transplant recipient were isolated ~:~ using density gradient centrifugation, cultu~ed with PHAfor 18 hours, incubated with monoclonal antibodies, four parameter data generate~ and analyzed as described in "Detailed Description".
The following results were obtain~d:
,~, (18 hr. ~ul~ur~. pHA
BiopsyTritiated Diagnos1sThvmidine % of Activated CorrelDting Uptake (72 hr Activated Leu-3a~ expressing a Activated Nor~al not done110,013 71 30 Patient A non-rejecting Z9,706 60 37 11 20 Patient A rejection211,867 73 39 26 Patlent A~* rej0ction170,953 66 10 39 Patient A rejection45,172 87 52 59 Percentage of ant;genic determinant positiYe lymphocytes which express greater than the preset minimai IL-2 receptor density.
This sample was drawn about 2 hours after the patien~ had received his immunosuppress;ve~medication.

,, From this study it can be seen that the present assay uses four parameter data analysis of stimulatd mononuclear c211s to obtain more detailed information concerning the immunoregulatory status of the mononuclear leukocyte immune system in less than 24 hours. The activation of lymphocyte subclasses after culturing with PHA for 18 hour is measured by determining the degree of IL-2 receptor activation antigen expression on these : subclasses. Tritiated thymodine uptake i5 determined after a 72 hour culture with PHA~ The results in Table IX
illustrate the correlation of activated Leu-2a~ with rejection and percent of activated ~eu-3a+ expres~ing a high density of IL-2 receptor with the acute sffects of immunosupressive therapy during a rejection episode. This information can be more readily correlated with an alter~d immunoregulatory ~tatus than the tritiated thymidine uptake assay.

Data generation and analy~iæ discussed in connection with the above embodiments of thi~ assay can be conducted using the Eollowing apparatu~. Fig. 14 is a : 3tylized functional and structural representation of : apparatus which may be u~ed in accordance wi~h th~
principle~ of the present invention to measure the four da~a parame~ers for each cell and to ~nalyze thi four parameter data to make ~ determination of the immunoregulatory status of the mononuclear leukocyte immune system, The apparatus is designed to yield an :; .

;.

accurate and reproducible measurement of activation ~; antigen expre~sion on subcla~ses of mononuclear ::~ leukocytes.
shown in Fig. 14, the apparatus oonsists of two major interrelated components and an interfacP module.
The general design of the fir~t component, a flow :: cytofluorometer (her~inafter FCFM~, is known in the prior : art and i~ briefly reviewed here.
: An aliquot of a prepared sample, which has been isolated, cultured with a standard stimulus and incubated wi~h specific monoclonal antibodies according to the methods presently disclosed, is received in machine intake port 97 for analysis. The sampl~ cells ~low through the flow ~ystem 105 including the flow chamber and channel 106 to the sen~ing zone 104 where each cell i~ illuminated with laser light from the la~er 100 through the focusing lens sy~tem 102. A~ i~ known in the art, the cell sample 3tream i5 carried in laminar fashion within a flowing fluid sheath/ to insure that a ~ingle cell wlll be illuminated in the sen~ing zon~ at a given time. Cell~ in liquid su~pen~ion are passed at a rapid rate (e.g. 2500 cell~ p~r ~econd) through the sQnsing zone~ Light i~
scattered to the ~orward light scatter photo~en~or 111 (e.g. ~hotodiode~ and amplifier) and to the orthogonal coll2ction lens 107. The orthogonal collect:ion lens is set:orthogonal to both the directio~ of the cell sample stream and axis of laser lightO Forward and orthogonal scattered light is collected in approximately a cone of ~` half-angle 20 degrees. The eleetronic ~ignal (hereinafter da~a signal) generated by th~ FLS photosensor i5 regulated .
i ,~
,~., .

z by way of the control 132 (e.g., ~ain control for an amplifier) in the interface 13G which can be modified by a control signal from the microcomputer system 140.
Light which is collected in the lens 107 i5 split and iltered by the series of beam splitters and filters 109. An example of a combination of beam splitters and filters i~ described in the "Detailed Description'i and is reiterated here as being exemplary, but not limiting, to the scope of the invention. Light of 10 wavelength less than 438 nm i5 reflected ~y a 488 nm dichroic splitter and enters the photos~nsor 115 (e.g.
photomultiplier tube with an associa~ed preamplifier) ~; which is regulated by the control 133 (e.g. an applied high voltage control for a photomul~iplier tube) in the interface 130. In thi~ example, this photosensor is used to detect orthogonal light scatter.
Light o~ wavelength greater than 488 nm i~
pas3ed by the 488 dichroic splitt~r and continue~ alony the orthogonal axis. The light passes a 515 nm inter~erence long pass filter and a 560 nm dichroic splitter. Light o~ wavelength greater than 560 nm i~
reflected to a 590 nm long pass filter and enters photosensor 118 which i3 re~ulated by the control 134 in :~ interface:l30. Light o~ wavelength less than 560 nm i~
pas~ed by the 560 nm dichrQic splitter to a 525 nm band pass filter and enters the photosen~or 1~0 which is regulated by the control 135 in the interface 13D. In this example the photosensor 118 is used to detect red : fluor2scence and the photosensor 120 is used to detect ; 30 green florescence. Each photosensor ~upplie~ an ,: .

' ~

~ ~$~i$~3~

electronic output signal, i.e. data signal. The photosensor controls 133, 134, 135 can be modified by : contro~ signals from the microcomputer ~ystem 140.
In addition to photosensor controls, the interface contain other data signal processing elements, e.g. pulse detectors or integrators, which may be used, in accordance with the abilities of those with ordinary skill the ar ~, to produce data signals of suitable characteristics for subsequent processing. The interface 130 also includes the amplifiers 136, 137 and 138 to amplify the data signal which is then supplied to the microcomputer sy~tem 140. In a preferred embodiment, these are logarithmio amplifiers. The inter~ace contains the contro~ signal lines 160, ;ncluding the control signal lines to the FC~M to control the laser 100 and the flow chamber and channel 106. By containing all important control and data ~i~nal proce~sing element~, the interface facili~ates maintenance and corrective sezvi~ing o~ the apparatu~.
~he microcomputer ~ystem 140 con~ists of a central proce~ing unit, memory, a terminal for operator nterface, a data storage devicet A to D converters or the incoming data siynals on the data signal lines 150 to A converters or the outgoing control ~ignals on the control signal lines 160 and the software to analyze the data signals and to ensure the accuracy and reproducibility of the measurements made by the FCFM 98.
The system's software contains routines to perform the ~ followiny functions: four parameter data storage for each ;~ 30 cell within threshold ranges pertinent to the analysis, , :

analysis of light scatter data parameters to determine the : class of each cell, compensation of fluorescence data parameters of each cell for ~he overlap of the emission spectra of different fluorophores, analysis of the S compensated fluorescence data parameter3 to determine the subclass and activation antigen density of each cell of a specified class, analysis of the degree of activation for each subclass to make a determination of the immunoregulatory status, report output, FCFM control, operator interface and system integra~ion/operation~
~ The system controller routine ensures the proper integration of the other software routines a~d overall operation of the system, including the storage and retrieval of data from the storaye device. It i5 ~ubstantially a disk operating system as is known in the art~ with modifications pertinent to this method and apparatus. The system controller routine communicate~
with the operator of the apparatus to accept data concerning the aliquot of the patient sample to be analyzed, this can include clinical data about the patient.
The ~torage routine will process th~ incoming data signals to det~rmine if the light scatter parameters of a c~ll are within threshold ranges se~ by the operator ~:~ 25 and store the four parameter data for that cell.
The light scatt2r analy~is routine will determine the class for each cell stor~d using preset areas of interest identifying certain classes. In a preerred embodiment, Eorward and orthogonal light scatter are used to make class determinations. If the cell is . ~ . .
" "" ' '~ ' .' ~, . ~

~ 3 :`

determined to be in an operator-specified class, further a~alysis is performed by the compen~ation and fluorescence analysis routines.
For those ~ells in a specified class as indicated by the light scatter analycis routine, the compensation routine corrects the fluorescence data parameters of the cell for the overlap of the emission spectra of the fluorophores which were conjugated ~o the monoclonal antibodies. In a preferred embodiment, the percentages are pre et and determined by the iterative process described in the "Detailed Description"~ The compensated fluorescence parameter data are then passed to the fluorescence analy~is routine. In an alternative .embodiment, the cQmpensation routine could be implemented with hardware as has been done in the prior art.
: The fluorescence analyzex collect~ the : compensated fluorescence parameter data for all cell~ of a specified cla~s as indicated by the light scatter analysis routin~. This routine identifies the subclass, enumerate~
th~ cells in the subc:Las~ and those cells in the subclass which ~re activated. This collected data i~ referred to as cumulative data and can be represented by histograms.
ln a preferr2d embodiment as describet in the "Detailed Descri~tion", a histogram of the amount of fluorescence indicative of antigenic determinants on the cell i5 ~- generated. The channel number, on the histogram~
representing the x-intercept of an approximation of the tangent to the positive slope of the curve generated is used as the minimal density necessary for a cell to qualify a~ a memher of the specified subclass. A similar procedure can be used to determine the maximal antigenic determinant density from the negative slope of the curve generated, alternatively, a default value set at th~
highest density measurable can be used. The cumulative data is then analyzed using theRe density boundaries t~
enumerate cells which are positive for the antiyenic de~erminant and can be assigned to the subclassO Using the channel number indlcative of cellular activation determined by the iterative proce~s as described in the I'Detailed Description'l, the activated cells belonging to the subclass are enumerated. Also, a range can be specified such that cells belonging to the subclass and having activation antigen expr~ssion within the r~nge can be enumerated. Th~ fluorescense analyzer can also monitor : 15 the degree of antigenic determinant modulation with actiuation or enumerate c~ with two speci~ic antigenic determinants of known den itie~. An alkernative within . the scope of thi~ invention i~ an add.itional, orthogonally : placed photo~ensor with a data signal conveying information on a third wavelength of fluoEe~cence to be used to identify distinct antigenic determinants~ This alternative can include an additional las~r with an associated ~ocu~ing lens system and delay circuit for any other ignal processing el~ment s~ooia~e~ with the additional photosensor, as i~ known in the art.
~ nalysis of the de~ree of activation for each subclas~ to assess the immunoregulatory status is done by the statistical analyzer routine~ The statistical analyzer routine receives information from the : 30 fluorescence analysis routine concerning class, quan~ity ' ' , .
, of cell~ in the subclass, quantity of activated cells in the subcla~s and other determinations, e.g. quantity of cells in the subclass expressing activation anti~en within a certain ran~e. The statistical analyzer routine uses this information to determine the level oE ~ctivation, e.g. percentage of a subclass which is activated, percentage of activated cells in a subclass expressing activation an~igens at a high den~ity, ratios of activated subclasses, combinations of these determinations, etc~
: 10 (~or illustrations, see the Examples above.) The report output routine generate a report with the ~ssessment of the immunoregulatory status as determined by the statistical analyzer routine. The repoxt can contain averaged result~ ~or normal individuals or patients similar to the one analyæed for Purther a~e~sment by the clinician.
To ensure accuracy and reproducibility of the :~ mea~urement~ made by the FCFM 9~ and therefore the re~ult~
produced by the apparatus, uniformly shaped control spheres wLth a pr~determined ~uantity of fluorophores bound to their ~urfaces can be analyzed by ~he appara~us.
The values o~ the four parameter data for the~e sph~res are predetermin~d. Therefore~ the light scat~er and fluorescence analysi~ routine~ can collec~ da~a on an ali~uot of a sample o~ control ~phere . The mean value for each parameter can be determined from this cumula~ive :~ four parameter data and ~ent to the FCFM control routine.
The FCFM control routine compares these value~ with the predetermined values. ~djustment~ o~ the FCFM 9~ and photosensor controls can be made by generating control ~ ' -.

signals such that accurate and reproducible measurement3 are made. This often involves an iterative process of rerunning the control spheres until there is no significant deviation from the predetermined values. This facilita~es the e~tablishment of preset areas of interest to be u ed to process patient samples. It should be noted that by including the control spheres in the aliquot of cells at a known concentration, analysis of these spheres while data is being collec~ed on the cells can assist in flow system clog detection.
The FCFM control routine also controls the operation of the FCFM 98 per the instruction~ of the system controller routine.
:~ The applications of thi~ apparatus include, but are not limited to, the Exampleq deqcribed above. The apparatus ensure3 the accuracy, reproducibility and ~imeliness o~ the result~ of the assay, these being nece~sary to as~i~t in the clinical diagnostic and therapeutic decision process. Also, the ability to detect ~0 clog~ in the flow sy~tem assi~ts in ensuring that there is minimal 109~ of the clinical ~ampleO
It i~ to be understood that the schematics of Fig. 14 are merely representa~ive of the unctional ~: aspect~ of the principles of the presen~ invention, and may be embodied in numerous alternative fashions in accordance with the abilities of one of ordinary skill in the art.

.

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Claims (71)

1. A method for assessing the immunoregulatory status of the immune system by generating and analyzing data on activated cells in select mononuclear cell subclasses from a sample of mono-nuclear cells cultured with a standard stimulus comprising:
a. isolating a sample which is substantially comprised of peripheral mononuclear cells;
b. culturing said sample of mononuclear cells with a standard stimulus for a period of time sufficient to allow for measurable cellular activation, as influenced by cell subclass interaction, to develop;
c. generating data on individual cells of said sample indicative of select mononuclear cell subclasses and cellular activation;
d. performing a first analysis of said data to identify and enumerate cells in said select mononuclear cell subclasses;
e. performing a second analysis of said data to identify and enumerate activated cells in said select mononuclear cell sub-classes; and f. performing a comparison of said enumerations of said first and second analyses to determine the degree of cellular activation in said select mononuclear cell subclasses, said degree being an assessment of the immunoregulatory status of the immune system.

2. A method as defined in Claim 1, wherein said standard stimulus is PHA,

3. A method as defined in Claim 1, wherein said data generated on individual cells of said sample is indicative of the helper/inducer T lymphocyte subclass and cellular activation.

4. A method as defined in Claim 1, wherein said data gener-ated on individual cells of said sample is indicative of the suppressor/cytotoxic T lymphocyte subclass and cellular activa-tion.

5. A method as defined in Claim 1, wherein said data gener-ated on individual cells of said sample is indicative of select mononuclear cell subclasses and activation antigen expression.

6. A method as defined in Claim 1, wherein said second analysis of said data is to enumerate activated cells in said select mononuclear cell subclasses, cells in said select mono-nuclear cell subclasses having greater than a preset minimal amount of cellular activation necessary to identify cells in said select mononuclear cell subclasses as being activated.

7. A method as defined in Claim 1, wherein said second analysis of said data is to identify and enumerate activated cells in said select mononuclear cell subclasses, cells in said select mononuclear cell subclasses having an amount of cellular activa-tion within a certain preset range.

8. A method as defined in Claim 1, wherein said immune system is the mononuclear leukocyte immune system.

9. A method for assessing the immunoregulatory status of the mononuclear leukocyte immune system by generating and analyz-ing flow cytofluorometric data on activated cells in select mono-nuclear cell subclasses from a sample of mononuclear cells cultured with a standard stimulus comprising:
a. isolating a sample which is substantially comprised of peripheral mononuclear cells;
b. culturing said sample with a standard stimulus for a period of time sufficient to allow for measurable cellular activa-tion, as influenced by mononuclear cell subclass interaction, to develop;
c. incubating said sample with fluorometrically distinguish-able fluorophore-conjugated monoclonal antibodies to identify select mononuclear cell subclass antigenic determinants and select cell surface activation antigens;
d. generating flow cytofluorometric data on individual cells of said sample indicative of select mononuclear cell subclasses and cellular activation;
e. performing a first analysis of said flow cytofluormetric data to identify and enumerate cells in said select mononuclear cell subclasses;
f. performing a second analysis of said flow cytofluoro-metric data to identify and enumerate activated cells in said select mononuclear cell subclasses; and g. performing a comparison of said enumerations of said first and second analyses to determine the degree of cellular activation in said select mononuclear cell subclasses, said degree being an assessment of the immunoregulatory status of the mono-nuclear leukocyte immune system.

10. A method as defined in Claim 9 wherein the standard stimulus is PHA.

11. A method as defined in Claim 9 wherein said sample is cultured with said standard stimulus, the culture being terminated within about 24 hours.

12. A method as defined in Claim 9 wherein said standard stimulus is PHA, the culture being terminated within about 18 hours.

13. A method as defined in Claim 9, wherein said fluoro-metrically distinguishable fluorophore-conjugated monoclonal anti-bodies include a monoclonal antibody which binds to an antigenic determinant associated with the helper/inducer T-lymphocyte sub-class.

14. A method as defined in Claim 9, wherein said fluoro-metrically distinguishable fluorophore-conjugated monoclonal anti-bodies include a monoclonal antibody include a monoclonal anti-body which binds to an antigenic determinant associated with the]
suppress or/cytotoxic T-lymphocyte subclass.

15. A method as defined in Claim 9, wherein said fluoro-metrically distinguishable fluorophore-conjugated monoclonal anti-bodies include a monoclonal antibody which binds to an antigenic determinant associated with the T-lymphocyte subclass.

16. A method as defined in Claim 9, wherein said fluoro-metrically distinguishable fluorophore-conjugated monoclonal anti-bodies include a monoclonal antibody which binds to the interleukin-2 receptor activation antigen.

17. A method as defined in Claim 9, wherein said flow cyto-fluorometric data include light scatter and fluorescence measure-ments.

18. A method as defined in Claim 9, wherein said second analysis of said flow cytofluorometric data is to enumerate activated cells in said select mononuclear cell subclasses, cells in said select mononuclear cell subclasses having greater than a preset minimal amount of activation antigen expression necessary to identify cells in said select mononuclear cell subclasses as being activated.

19. A method as defined in Claim 9, wherein said second analysis of said flow cytofluorometric data is to identify and enumerate activated cells in said select mononuclear cell sub-classes, cells in said select mononuclear cell subclasses having an amount of activation antigen expression within a certain preset range.

20. A method as defined in Claim 9 wherein said flow cyto-fluorometric data is corrected for nonspecific binding of fluorophore-conjugated monoclonal antibodies.

21. A method for assessing the immunoregulatory status of the mononuclear leukocyte immune system by generating and analyz-ing cumulative four parameter data on activated cells in select mononuclear cell subclasses from one or more aliquot(s) of mono-nuclear cells cultured with a standard stimulus comprising:
a. isolating a sample which is substantially comprised of peripheral mononuclear cells;
b. culturing said sample with a standard stimulus for a period of time sufficient to allow for measurable cellular activa-tion, as influenced by mononuclear cell subclass interaction, to develop;
c. incubating one or more aliquots of cells from said sample with a first fluorophore-conjugated monoclonal antibody specific for a select mononuclear cell subclass antigenic determinant and a second fluorometrically distinguishable fluorophore-conjugated monoclonal antibody specific for a select cell surface activation antigen;
d. generating four parameter data from measurements of forward light scatter, orthogonal light scatter, fluorescence of a first fluorophore and fluorescence of a second fluorophore on individual cells from each aliquot of cells using flow cytofluoro-metric techniques;
e. performing a first analysis of the cumulative four para-meter data for each aliquot of cells to determine the minimal density of antigenic determinant expression on individual mono-nuclear cells necessary to identify said individual mononuclear cells as a member of said select mononuclear cell subclass;
f. performing a second analysis of the cumulative four para-meter data for each aliquot of cells to enumerate mononuclear cells having greater than the minimal density of said antigenic determinant expression;
g. performing a third analysis of the cumulative four para-meter data for each aliquot of cells to enumerate activated cells in said select mononuclear cell subclass, cells in said select mononuclear cell subclass having greater than a preset minimal density of activation antigen expression necessary to identify cells in said select mononuclear cell subclass as being activated, and h. performing a comparison of said enumerations of said second and third analyses for each aliquot of cells to determine the degree of cellular activation in said select mononuclear cell subclass, said degree being an assessment of the immunoregulatory status of the mononuclear leukocyte immune system.

22. A method as defined in Claim 21 wherein the standard stimulus is PHA.

23. A method as defined in Claim 21 wherein said sample is cultured with said standard stimulus, the culture being terminated within about 24 hours.

24. A method as defined in Claim 21 wherein said standard stimulus is PHA, the culture being terminated within about 18 hours.

25. A method as defined in Claim 21 wherein said fluorophore-conjugated monoclonal antibody specific for select mononuclear cell subclass binds to an antigenic determinant associated with the helper/inducer T-lymphocyte subclass.

26. A method as defined in Claim 21 wherein said fluorophore-conjugated monoclonal antibody specific for select mononuclear cell subclass binds to an antigenic determinant associated with the suppressor/cytotoxic T-lymphocyte subclass.

27. A method as defined in Claim 21 wherein said fluorophore conjugated monoclonal antibody specific for select mononuclear cell subclass binds to an antigenic determinant associated with the T-lymphocyte subclass.

28. A method as defined in Claim 21 wherein said fluorophore-conjugated monoclonal antibody specific for a select cell surface activation antigen binds to the interleukin-2 recep-tor activation antigen.

29. A method as defined in Claim 21 wherein said first fluorophore is fluorescein-isothiocyanate and said second fluoro-phore is phycoerythrin.

30. A method as defined in Claim 21 wherein said first fluorophore is phycoerythrin and said second fluorophore is fluorescein-isothiocyanate.

31. A method as defined in Claim 21 wherein said first, second and third analyses of the cumulative four parameter data for each aliquot of cells identify cells of a select mononuclear cell class using a preset light scatter area of interest.

32. A method as defined in Claim 21 wherein said analysis of the cumulative four parameter data for each aliquot of cells identifies cells of a select mononuclear cell class using a preset light scatter area of interest adjusted based on analysis of cumulative, forward and orthogonal light scatter data generated using flow cytoflurometric techniques for an aliquot of cells obtained before culturing said sample.

33. A method as defined in Claim 21 wherein said third analysis of the cumulative four parameter data for each aliquot of cells is to identify and enumerate activated cells in said select mononuclear cell subclass, cells in said select mononuclear cell subclass having activation antigen expression within a certain preset specified density range.

34. A method as defined in Claim 21 wherein an aliquot of cells is incubated with a said first fluorophore-conjugated mouse-IgG1 control monoclonal antibody and a said second fluorometrical-ly distinguishable fluorophore-conjugated mouse-IgG1 control mono-clonal antibody, to quantitate nonspecific binding of said mono-nuclear cells.

35. A method as defined in Claim 21 wherein a first aliquot is incubated with said fluorophore-conjugated monoclonal antibody specific for select mononuclear cell subclass binds to an anti-genic determinant associated with the helper/inducer T-lymphocyte subclass and a second aliquot is incubated with said first fluorophore-conjugated monoclonal antibody specific for select mononuclear cell subclass binds to an antigenic determinant associated with the suppressor/cytotoxic T-lymphocyte subclass.

36. A method for assessing the immunoregulatory status of the mononuclear leukocyte immune system by generating and analyz-ing cumulative four parameter data on activated cells in select mononuclear subclasses from one or more aliquot(s) of mononuclear cells cultured with a standard stimulus comprising:
a. isolating a sample which is substantially comprised of peripheral mononuclear cells;

b. dividing said sample into separate portions of cells;
c. incubating one or more aliquots of a first portion of cells with a first fluorophore-conjugated monoclonal antibody specific for a select mononuclear cell subclass antigenic determi-nant and a second fluorometrically distinguishable fluorophore-conjugaged monoclonal antibody for a select cell surface activa-tion antigen;
d. culturing a second portion of cells with a standard stimulus for a period of time sufficient to allow for measurable cellular activation, as influenced by mononuclear cell subclass interaction, to develop;
e. incubating one or more aliquots of said second portion of cells with a first fluorophoro-conjugated monoclonal antibody specific for a select mononuclear cell subclass antigenic determi-nant and a second fluorometrically distinguishable fluorophore-conjugated monoclonal antibody specific for a select cell surface activation antigen;
f. generating four parameter data from measurements of forward light scatter, orthogonal light scatter, fluorescence of a first fluorophore and fluorescence of a second fluorophore on individual cells from each aliquot of cells using flow cytofluoro-metric techniques;
g. performing a first analysis of the cumulative four para-meter data for each aliquot of cells to determine the minimal density of antigenic determinant expression on individual mono-nuclear cells necessary to identify said individual mononuclear cells as a member of said select mononuclear cell subclass;

h. performing a second analysis of the cumulative four para-meter data for each aliquot of cells to enumerate mononuclear cells having greater than the minimal density of said antigenic determinant expression;
i. performing a third analysis of the cumulative four para-meter data for each aliquot of cells to enumerate activated cells in said select mononuclear cell subclass, cells in said select mononuclear cell subclass having greater than a preset minimal density of activation antigen expression necessary to identify cells in said select mononuclear cell subclass as being activated;
and j. performing a comparison of said enumerations of said second and third analyses for each aliquot of cells to determine the degree of cellular activation in said select mononuclear cell subclass, said degree being an assessment of the immunoregulatory status of the mononuclear leukocyte immune system.

37. A method as defined in Claim 36 wherein the standard stimulus is PHA.

38. A method as defined in Claim 36 wherein the sample is cultured with said standard stimulus, the culture being terminated within about 24 hours.

39. A method as defined in Claim 36 wherein said standard stimulus is PHA, the culture being terminated within about 18 hours.

40. A method as defined in Claim 36 wherein said fluorophore-conjugated monoclonal antibody specific for select mononuclear cell subclass binds to an antigenic determinant associated with the helper/inducer T-lymphocyte subclass.

41. A method as defined in Claim 36 wherein said fluorophore-conjugated monoclonal antibody specific for select mononuclear cell subclass binds to an antigenic determinant associated with the suppressor/cytotoxic T-lymphocyte subclass.

42. A method as defined in Claim 36 wherein said fluorophore-conjugated monoclonal antibody specific for select mononuclear cell subclass binds to an antigenic determinant associated with the T-lymphocyte subclass.

43. A method as defined in Claim 36 wherein said fluorophore-conjugated monoclonal antibody specific for a select cell surface activation antigen binds to the interleukin-2 recep-tor activation antigen.

44. A method as defined in Claim 36 wherein said first fluorophore is fluorescein-isothiocyanate and said second fluoro-phore is phycoerythrin.

45. A method as defined in Claim 36 wherein said first fluorophore is phycoerythrin and said second fluorophore is fluorescein-isothiocyanate.

46. A method as defined in Claim 36 wherein said first, second and third analyses of the cumulative four parameter data for each aliquot of cells identify cells of a select mononuclear cell class using a preset light scatter area of interest.

47. A method as defined in Claim 36 wherein said analysis of the cumulative four parameter data for each aliquot of cells identifies cells of a select mononuclear cell class using a preset light scatter area of interest adjusted based on analysis of cumulative, forward and orthogonal light scatter data generated using flow cytoflurometric techniques for an aliquot of said first portion of cells.

48. A method as defined in Claim 36 wherein said third analysis of the cumulative four parameter data for each aliquot of cells is to identify and enumerate activated cells in said select mononuclear cell subclass, cells in said select mononuclear cell subclass having activation antigen expression within a certain preset specified density range.

49. A method as defined in Claim 36 wherein an aliquot of cells is incubated with a said first fluorophore-conjugated mouse-IgG1 control monoclonal antibody and a said second fluorometrical-ly distinguishable fluorophore-conjugated mouse-IgG1 control mono-clonal antibody, to quantitate nonspecific binding of said mono-nuclear cells.

50. A method as defined in Claim 36 wherein a first aliquot is incubated with said first fluorophore-conjugated monoclonal antibody specific for select mononuclear cell subclass which binds to an antigenic determinant associated with the helper/inducer T-lymphocyte subclass and a second aliquot is incubated with said first fluorophore-conjugated monoclonal antibody specific for select mononuclear cell subclass which binds to an antigenic determinant associated with the suppressor/cytotoxic T-lymphocyte subclass.

51. A clinical method for assessing and monitoring the effects on the immunoregulatory status of the mononuclear leuko-cyte immune system of a patient by generating and analyzing cumulative four parameter data on activated cells in select mono-nuclear cell subclasses from one or more aliquot(s) of mononuclear cells cultured with a standard stimulus comprising:
a. isolating a sample which is substantially comprised of peripheral mononuclear cells;
b. culturing said sample with a standard stimulus for a period of time sufficient to allow for measurable cellular activa-tion, as influenced by mononuclear cell subclass interaction, to develop;
c. incubating one or more aliquots of cells from said sample with a first fluorophore-conjugated monoclonal antibody specific for a select mononuclear cell subclass antigenic determinant and a second fluorometrically distinguishable fluorophore-conjugated monoclonal antibody specific for a select cell surface activation antigen;
d. generating four parameter data from measurements of forward light scatter, orthogonal light scatter, fluorescence of a first fluorophore and fluorescence of a second fluorophore on individual cells from each aliquot of cells using flow cytofluoro-metric techniques;
e. performing a first analysis of the cumulative four para-meters data for each aliquot of cells to determine the minimal density of antigenic determinant expression on individual mono-nuclear cells necessary to identify said individual mononuclear cells as a member of said select mononuclear cell subclass;
f. performing a second analysis of the cumulative four parameter data for each aliquot of cells to enumerate mononuclear cells having greater than the minimal density of said antigenic determinant expression;
g. performing a third analysis of the cumulative four para-meter data for each aliquot of cells to enumerate activated cells in said select mononuclear cell subclass, cells in said select mononuclear cell subclass having greater than a preset minimal density of activation antigen expression necessary to identify cells in said select mononuclear cell subclass as being activa-ted;
h. performing a comparison of said enumerations of said second and third analyses for each aliquot of cells to determine the degree of cellular activation in said select mononuclear cell subclass, said degree being an assessment of the immunoregulatory status of the mononuclear leukocyte immune system of said patient;
and i. initiating, modifying or maintaining immunomodulation therapy for said patient based on the assessment of said immuno-regulatory status so as to alter or maintain said immunoregulatory status.

52. A method as defined in Claim 51 wherein the patient has received an organ transplant.

53. A method as defined in Claim 51 wherein the immuno-modulation therapy is cyclosporin.

54. A method as defined in Claim 51 wherein the immuno-modulation therapy is a glucocorticoid.

55. A method as defined in Claim 51 wherein the standard stimulus is PHA.

56. A method as defined in Claim 51 wherein the sample is cultured with said standard stimulus, the culture being terminated within about 24 hours.

57. A method as defined in Claim 51 wherein said standard stimulus is PHA, the culture being terminated within about 18 hours.

58. A method as defined in Claim 51 wherein said fluorophore-conjugated monoclonal antibody specific for select mononuclear cell subclass binds to an antigenic determinant associated with the helper/inducer T-lymphocyte subclass.

59. A method as defined in Claim 51 wherein said fluorophore-conjugated monoclonal antibody specific for select mononuclear cell subclass binds to an antigenic determinant associated with the suppressor/cytotoxic T-lymphocyte subclass.

60. A method as defined in Claim 51 wherein said fluorophore-conjugated monoclonal antibody specific for select mononuclear cell subclass binds to an antigenic determinant associated with a T-lymphocyte subclass.

61. A method as defined in Claim 51 wherein said fluorophore-conjugated monoclonal antibody specific for a select cell surface activation antigen binds to the interleukin-2 recep-tor activation antigen.

62. A method as defined in Claim 51 wherein said first fluorophore is fluorescein-isothiocyanate and said second fluoro-phore is phycoerythrin.

63. A method as defined in Claim 51 wherein said first fluorophore is phycoerythrin and said second fluorophore is fluorescein-isothiocyanate.

64. A method as defined in Claim 51 wherein said first, second and third analyses of the cumulative four parameter data for each aliquot of cells identify cells of a select mononuclear cell class using a preset light scatter area of interest.

65. A method as defined in Claim 51 wherein said analysis of the cumulative four parameter data for each aliquot of cells identifies cells of a select mononuclear cell class using a preset light scatter area of interest adjusted based on analysis of cumulative, forward and orthogonal light scatter data generated using flow cytoflurometric techniques for an aliquot of cells obtained before culturing said sample;

66. A method as defined in Claim 51 wherein said third analysis of the cumulative four parameter data for each aliquot of cells is to enumerate activated cells in said select mononuclear cell subclass, cells in said select mononuclear cell subclass having activation antigen expression within a certain preset specified density range.

67. A method as defined in Claim 51 wherein an aliquot of cells is incubated with a said first fluorophore-conjugated mouse-IgG1 control monoclonal antibody and a said second fluorometrical-ly distinguishable fluorophore-conjugated mouse-IgG1 control mono-clonal antibody, to quantitate nonspecific binding of said mono-nuclear cells.

68. A method as defined in Claim 51 wherein a first aliquot is incubated with said first fluorophore-conjugated monoclonal antibody specific for select mononuclear cell subclass binds to an antigenic determinant associated with the helper/inducer T-lympho-cyte subclass and a second aliquot is incubated with said first fluorophore-conjugated monoclonal antibody specific for select mononuclear cell subclass which binds to an antigenic determinant associated with a suppressor/cytotoxic T-lymphocyte subclass.

69. Apparatus for assessing the immunoregulatory status of the mononuclear leukocyte immune system of a sample consisting substantially of peripheral mononuclear cells cultured with a standard stimulus and incubated with a first fluorophore-conjugated monoclonal antibody specific for a select mononuclear cell subclass antigenic determinant and a second fluorophore-conjugated monoclonal antibody specific for a select cell surface activation antigen comprising:
a. flow means for passing cells of said sample rapidly and substantially one at a time through a sensing zone;
b. means for stimulating fluorescent activity of said first and second fluorophores passing through said sensing zone;
c. photosensing means for detecting light scatter from said sensing zone in at least one predetermined direction indicative of cell morphology:
d. photosensing means for sensing fluorescence from said first fluorophore in said sensing zone, indicative of said select cell subclass;
e. photosensing means for sensing fluorescence from said second fluorophore in said sensing zone, indicative of said activation antigen; and f. means connected to said photosensing means for determin-ing the quantity of cells in said select cell subclass and the quantity of cells expressing greater than a preset minimal density of activation antigen.

70. Apparatus for assessing the immunoregulatory status of the mononuclear leukocyte immune system of a sample consisting substantially of peripheral mononuclear cells cultured with a standard stimulus and incubated with a first fluorophore-conjugated monoclonal antibody specific for a select mononuclear cell subclass antigenic determinant and a second fluorophore-conjugated monoclonal antibody specific for a select cell surface activation antigen comprising:
a. a cytofluorometric flow means for passing cells of said sample, rapidly and substantially one at a time through a given area;
b. a laser means for stimulating fluorescent activity of said first and second fluorophores in said given area;
c. first photosensing means for detecting light scatter from said given area in a first predetermined direction;
d. second photosensing means for detecting light scatter from said given area in a second predetermined direction;
e. third photosensing means for sensing fluorescence from said first fluorophore in said given area;
f. fourth photosensing means for sensing fluorescence from said second fluorophore in said given area;
g. means connected to said first and second photosensing means for determining cell class on the basis of said light scatter parameters; and h. means connected to said third and fourth photosensing means for determining the quantity of cells in said select cell subclass and the quantity of cells expressing greater than a pre-set minimal density of activation antigen.

71. The apparatus of Claim 70 wherein the sample to be analyzed contains control spheres with predetermined light scatter and fluorescence measurements and the apparatus further includes means for periodically adjusting the photosensing means to mini-mize the amount of deviation of the actual light scatter and fluorescence measurements of said control spheres from the pre-determined measurements.