FIELD OF THE INVENTION

This invention relates to a composition for fixing and staining a cell sample. The invention also relates to cell sample containers (such as specimen collection containers) comprising such a composition. In particular, the invention relates to the use of such a composition or container to stabilise a cell sample for transportation so that it may be directly analysed at a time or location distant from that of its collection.

BACKGROUND TO THE INVENTION

Concerted action is needed to help people in the underprivileged areas of the globe where HIV infection is currently spreading unabated. At last, after long international disputes, the concept of providing anti-retroviral drugs based on generic prices is being accepted. It is essential, however, that these treatments are accompanied by appropriate patient monitoring to diminish inefficient drug use and the development of drug resistant virus strains.

The monitoring of the patients' immune status can be efficiently done by immunological tests, such as the CD4-assay. There have been efforts to produce simple immunological tests that might operate at a village- or small hospital level (e.g. by using dipstick and dried blood spot technology), but so far these have not proved satisfactory. The best method for analysing the expression of cell surface markers is using a flow cytometric assay which needs sophisticated equipment and trained personnel to perform.

It is thus currently necessary to transport cell samples from the site of collection to a laboratory which has the equipment and expertise needed for analysis. WO 97/45729 describes a cellular fixative which is capable of stabilising cells for 7-14 days enabling the transportation of samples for longer distances. Blood samples can thus be collected and fixed at small centres before transfer to regional laboratories (such as those associated with district hospitals) for analysis. At the regional laboratory, one or more antibody(ies) is added and the sample analysed, for example by flow cytometry.

The addition of antibodies at the regional laboratory introduces the potential for pipetting errors at this stage and the need for antibody standardisation between laboratories.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that, unlike other fixatives, the fixative described in WO 97/45729 (which is commercially available as TransFix™) has the capacity to be mixed directly with an antibody without causing the antibody to denature. This open up the possibility of pre-mixing the fixative and antibody to simplify sample handling.

It is thus possible to provide a container, designed for transport, which contains a mixture of fixative and antibody in a fixed stable fluid volume or in a lyophilised form. In addition, the same container or tube can optionally contain an anticoagulant. This is advantageous because usually the analysis is for studying cells in suspension in anticoagulated blood. A sample such as a blood sample may then be directly added to the container, causing the sample cells to remain in suspension while being fixed and stained. After transport, the sample may be directly analysed without the need for addition of further reagents (such as antibody).

This system has numerous advantages:

(i) it is easier to standardise antibody reagents between analysis laboratories

(ii) the potential for pipetting errors and pipetting variations between different laboratories is reduced

(iii) the fixative also stabilises the antibody, minimising the possibility for variations in antibody quality

(iv) since the travelling time of the samples can be considerably extended, samples can be sent to larger laboratories, where the technological expertise for proper investigation is available.

The system also has advantages for more general applications. Firstly, it enables the assay to be "speeded up". For example, for CD34 stem cell assays, where the results are needed quickly, the availability of apre-mixed vial speeds up this part of the assay. Use of the system would also allow the sample to be retested later if technical problems occurred.

In a first aspect, the present invention provides a composition for mixing with a cell sample to fix and stain the cell sample, which comprises a mixture of a fixative and one or more antibodies. The composition may be provided, for example, as a fluid or as a lyophilized product, possibly deposited inside a tube.

The fixative solution (before lyophilisation or after reconstitution) comprises a sterile buffered aqueous solution comprising an aliphatic aldehyde, at a molar concentration of 0.15 to 3.4M, one or more heavy metal salts, at a total molar concentration of 0.2xl0 ^"3 M to 0.2M, the solution having a pH in the range of 6.8 to 8.0.

Lyophilized compositions may be made by drying or freeze-drying in a vacuum using standard techniques.

The or each antibody may recognise a cell surface marker such as a marker on an immune cell.

For use to monitor HIV infection, the antibody may recognise, for example CD4, CD8, CD38 or CD45.

The composition may also comprise an anticoagulant.

In a second aspect, the present invention provides a container for receiving a cell sample which comprises a composition according to the first aspect of the invention.

The container may be suitable for use in a flow cytometer. For example, the container may be a tube compatible with the flow cytometer chosen for analysis purposes, so that the container may be directly transferred to the flow cytometer for analysis.

Conveniently, the container may be adapted to facilitate addition of a predetermined volume of cell sample. If for example the container is marked with a "fill-line" to indicate a particular volume this would aid the addition of the required amount of cell sample (such that the combined volume of sample + composition reaches the fill line). The "fill-line" could act as a "cross-checking" mechanism to check for pipetting errors.

If the container is a conventional flow cytometry tube, the fill line may, for example, be at 0.5, 1 or 1.5 ml. If the container is a multi-well plate (for example a 96-well plate) the fill line may, for example, be at 50, 100 or 150 μl.

The third aspect of the invention relates to a kit which comprises at least one container according to the second aspect of the invention. The kit may comprise a plurality of such containers.

The new combined flow cytometric method for HIV monitoring may be performed using a "one-tube" or a "two-tube" system.

For use with the "one-tube" system, the kit may comprise a container according to the second aspect of the invention comprising a mixture of three or four antibodies for three or four-colour immunofluorescence. Suitable antibody mixtures include:

CD3, CD4, CD8 and CD45;

CD3,CD19,CD16 and CD56; and

CD4,CD8,CD38 andCD45, and other similar combinations. In a preferred embodiment the kit comprises CD4, CD8 and CD38 (and optionally CD45) antibodies.

For use with the "two-tube" system the kit may comprise: a) a first container comprising two antibodies; and b) a second container comprising two antibodies for two-colour immunofluorescence.

For example, the kit may comprise: a) a first container comprising CD4 and CD45 antibodies; and b) a second container comprising CD8 and CD38 antibodies

or similar antibody combinations.

In a fourth aspect, the present invention provides a method for preparing a cell sample for analysis which comprises the step of mixing the cell sample with a composition according the first aspect of the invention. The mixing may advantageously be achieved by directly adding the cell sample to the container.

hi a fifth aspect, the present invention provides a method for analysis of a specimen which comprises the steps of

(i) obtaining the specimen;

(ii) preparing the specimen for analysis by a method according to the fourth aspect of the invention; and

(iii) analysing the prepared specimen.

If the specimen is a blood sample, it may undergo hematological analysis. The method of the fourth aspect of the invention is particularly suitable for preparing a sample for analysis by an immunofluorescent method, such as flow cytometry. The analysis may involve examination of one or more haematological parameters, such as for example red blood cell counts, haemoglobin, haematocrit, white blood cell counts, white blood cell differentials and platelet counts.

Since the fixative fluid is capable of stabilising the stained cell sample for prolonged periods, steps (ii) and (iii) of the method of the fifth aspect of the invention maybe carried out at different sites and/or at different times.

The method of the fifth aspect of the invention may be used, for example, to (i) diagnose a disease; (ii) monitor the progression of a disease; (iii) monitor the therapy of a disease;

(iv) select an appropriate treatment program for a disease; and/or (v) investigate the epidemiology of the disease.

The disease may be a viral infection, such as HIV infection.

In a sixth aspect, the present invention provides a method for preparing a composition according to the first aspect of the invention by mixing a fixation fluid with an antibody. The fixative fluid and antibody are mixed in the absence of a cell sample. The cell sample may be added to the pre-mixed composition at a later stage.

DESCRIPTION OF THE FIGURES

Figure 1 shows the staining of a normal blood preparation with a CD4 monoclonal antibody that has been labelled with Alexa-488-dye. The sample has been stained by saturating amounts of antibody (5ug/ml-Fig 1 A), and its 100-fold (Fig IB and E) and 200-fold (Fig 1 C and F) dilution. The same sample has also been stained with the same antibody dilutions, kept in the presence of Transfix for 14 days (Fig 1 E and F). Fig ID shows the negative control.

The following detailed description elaborates on certain elements of the above aspects of the invention. These elements are presented under separate section headings. However, it is to be understood that the teachings under each section heading are not necessarily limited to that particular section heading.

DETAILED DESCRIPTION

COMPOSITION

The first aspect of the invention relates to a composition capable of fixing and staining a cell sample.

The concept of fixing and staining cells and tissue sections for immunological or histological analysis has been known for many decades. Chapter 10 in "Antibodies - A Laboratory Manual" (1988, Cold Spring Harbor Laboratory) summarises the various steps in antibody staining techinques, namely:

1) cell or tissue preparation

2) fixation

3) antibody binding

4) detection.

The term "fix" in the context of the present invention means to stabilise the cell sample such that the antigen is retained in such a form that it can be recognised by the antibody and to maintain cell structure to the extent that the cell can be characterised by virtue of their expression of antigen.

The term "stain" means to bring the cell sample into contact with the antibody such that the antibody my bind antigen, enabling the antigen to be detected.

The composition of the present invention comprises a fixation fluid and one or more antibodies. The ratio of volume of the or each antibody to fixation fluid may be , for example, between, 1:5 and 1:50. Usually the ratio is between 1:10 and 1:30, such as 1:20. For example, lOOul composition, may comprise 90ul fixation fluid, 5ul of a first antibody and 5ul of a second antibody. These fluids may be transformed into lyophilized form in order to prevent the dilution of the blood samples and therefore interfere with the absolute counts of cells.

FIXATIVE

The composition of the present invention maybe added to a sample collection tube, ready to receive a cell sample. In this context, the term "specimen collection fluid" and "fixation fluid" are interchangeable.

When biological specimens such as blood or bone marrow samples are analysed, it is often desirable to stabilise the specimens to enable storage and/or transport before analysis.

WO 97/45729 describes a specimen collection fluid which offers a number of advantages to those which were previously known in the art.

Before WO 97/45729, stabilisation fluids required the collection of anticoagulated blood and then the addition of ImI stabilisation fluid to ImI of the anticoagulated blood specimen, making the results of the subsequent immunohaematological analysis very difficult to interpret.

The fixation fluid of WO 97/45729 can be used in much smaller volumes, as well as in a lyophilized form. The addition of small amounts of the specimen collection fluid, usually around one in 100 parts, does not introduce a significant dilution factor which can affect the absolute value calculations.

Using the specimen collection fluid described in WO97/45729, immunohaematological analysis can be performed upon peripheral blood after more than 5 days following collection, without substantial deterioration in the antigen or cellular integrity. With this specimen collection fluid it has been found that the leucocyte and platelet antigens CD3, CD4, CD5, CD8, CDlO, CD13, CD16, CD14, CD 19, CD20, CD33, CD34, HLA-DR, and CD45, and also the haematological parameters normally measured, for example, white cell count, red cell count, platelet count, white cell differential and haemoglobin, can remain substantially stable during this period. Because the white cell count remains substantially constant throughout, this assists in the determination of absolute counts of cells that express the CD4 or CD8 or other antigens on their surface. RNA can be extracted from specimens for up to at least 5 days after collection, for example, for PCR analytical techniques.

The specimen collection fluid of W097/45729 significant benefits in the management of disorders such as AIDS. The peripheral blood parameters remain substantially stable, facilitating the transportation of specimens over long distances or allowing retention of specimens until times which are convenient for analysis.

The fixation fluid of WO 97/45729 which used in the present invention is a sterile buffered aqueous solution comprising the following:

(i) an aliphatic aldehyde, at a molar concentration of 0.15 to 3.4M; and

(ii) one or more heavy metal salts, at a total molar concentration of 0.2x10 ^"3 M to 0.2M. The solution has a pH in the range of 6.8 to 8.0.

The fixation fluid may be prepared by a person skilled in the art, but is also made by UK NEQAS under the name TransFix™. Jani et al (J. Imm. Meth. (2001) ρl45-154) describe the use of the TransFix™ with whole blood, finding it is capable of stabilising absolute CD4+ counts for at least 10 days at certain concentrations.

The aliphatic aldehyde used in the fixation fluid can be any suitable aliphatic aldehyde, but is preferably formaldehyde, and most preferably paraformaldehyde. To assist in dissolving the aldehyde in the aqueous solution the solution can be warmed if necessary, but the temperature should not be allowed to rise about 5O ⁰ C.

Suitable heavy metal salts are those of metals having complexing properties and having an atomic weight greater than 20, for example, transition metals, particularly transition metals of groups IVA to VIIA of the Periodic Table, for example, manganese, chromium, molybdenum, vanadium and titanium, group IB, for example, copper, and group IVB, for example, tin. Group VIA and group VIIA, transition metals, and especially chromium and manganese, are particularly preferred. Very good results have been obtained using manganese salts which have the advantage for some purposes that they have colourless solutions, and especially, mixtures of manganese and chromium salts.

Any suitable water soluble salts of such heavy metals may be used, especially inorganic acid salts, for example, sulphates, and particularly, chlorides. Particularly good results have been obtained using chromium and manganese compounds, for example, chromium salts such as chromic chloride CrCl ₃ , and manganese salts such as manganese chloride McCl ₂ , and these are the preferred metal salts for use in the present invention.

Any suitable anticoagulant can be used, although EDTA salts are preferred, for example, alkali metal salts, such as, di-potassium ethylenediaminetetraacetic acid (K ₂ EDTA) and tri-potassium ethylenediaminetetraacetic acid (K ₃ EDTA). Other anticoagulants which can be used include citrate/phosphate/dextrose/adenine (CPDA).

Preferred fixation fluids in accordance with the invention comprise aqueous solutions of paraformaldehyde, manganese and chromium chloride, and an anticoagulant. Preferably, the weight ratio of manganese to chromium is in the range of from 100:1 to 50:1, for example about 75:1.

The aqueous solution preferably comprises from 0.15 moles to 1.0 moles of the aliphatic aldehyde, from 0.2 x 10 ^"3 M to 0.1 or 0.2 moles of the heavy metal salts, and

from 0.27 to 0.45 moles of the anticoagulant. The aqueous solution preferably has a pH in the range of 7.2 to 7.6, for example, about 7.4.

The fixation fluid preferably also comprises one or more cell nutrients, for example, dextrose, adenosine tri-phosphate and inosine, and in amounts of respectively up to about 2.5M, 0.05M and 0.1M, and glycolitic pathway precursors, for example, di- hydroxy acetone and 2,3-diphosphoglycerol. Tri-sodium citrate and sodium chloride are, however, preferably omitted.

The fixation fluid preferably also comprises one or more antibiotics, to prevent bacterial growth which may otherwise occur, especially with nutrients present. Antibiotics, for example, chloramphenicol and neomycin sulphate, have been found to be suitable, in amounts of up to about 0.015M and 0.005M respectively.

The fixation fluid preferably also comprises a platelet stabiliser, for example magnesium chloride or iodoacetamide or its derivatives.

The fixation fluid may be made up comprising a sterile aqueous solution comprising the following components by weight:

paraformaldehyde 1.5 Molar Manganese chloride 0.125 Molar K ₂ EDTA 0.37 Molar

The fixation fluid may be made up comprising a sterile aqueous solution comprising the following components by weight:

paraformaldehyde 0.33 Molar Dextrose (D-glucose) 2.22 Molar Adenosine tri-phosphate 0.036 Molar Inosine 0.075 molar

Chloramphenicol 0.012 Molar

Neomycin Sulphate 0.0044 Molar

Chromium (III) Chloride 0.019 Molar

Manganese Chloride 0.025 Molar

Magnesium Chloride 0.52 Molar

The pH may be adjusted to about 7.4.

The fixation fluid can be used freshly made, or, if preferred, can be allowed to stand before use. It has been observed with some solutions of chromium compounds that the freshly made solutions give rise to the formation of a precipitate which it is believed may be a chromium hydroxy polymeric species. The precipitate is preferably filtered off from the solution before use. The formation of the precipitate will, of course, lower the concentration of heavy metal ions in the solution, and if this should occur, an analysis of the solution should be carried out to determine whether the concentration of the heavy metal salt is still within the preferred range.

ANTIBODY

As used herein, the term "antibody" includes a whole immunoglobulin molecule or a part thereof or a bioisostere or a mimetic thereof or a derivative thereof or a combination thereof. Examples of a part thereof include: Fab, F(ab)' _2; and Fv. Examples of a bioisostere include single chain Fv (ScFv) fragments, chimeric antibodies and bifunctional antibodies.

The term "mimetic" relates to any chemical which may be a peptide, polypeptide, antibody or other organic chemical which has the same binding specificity as the antibody.

The term "derivative" as used herein includes chemical modification of an antibody. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group.

The antibody may be monoclonal or polyclonal. The "antibody" may in fact be a pooled solution comprising a plurality of monoclonal antibodies directed to the same antigen.

For resource-poor situations generic monoclonal antibodies are becoming available from international agencies and/or government related quality assured organisations. Such generic monoclonal antibodies have been made and released to such organisations for CD4 (RFT4) and CD8 (RFT8) for use in HIV testing (Bofill et al (1992) Clin Exp Immunol 88: 243-252). Various CD45 reagents are known such as 2Dl (made by Beverley et al, Tumour Immunology Unit of the Imperial Cancer Research Fund; see Beverley et al. (1980) as above).

CD ANTIGEN MARKERS

The identification of membrane "markers" on immune system cells has been instrumental in the identification and characterization of functional cell types. Plasma membrane molecules unique to distinct functional cell types were identified by making antibodies to intact cells and studying the ability of those antibodies to bind to or inhibit the activities of different cells. An international CD (Cluster of Differentiation) classification system ensures that every antibody identified as anti-CD4, for example, binds the same molecule (not necessarily the same epitope).

Some common cell surface markers, for example BCR (membrane Ig) and TCR, have not been given CD designations, while other markers (B220 on B cells) have both a common name and a CD designation. A list of CD designations can be found at http://www.researchd.com/rdi cdabs/cdindex .htm Many are listed in Janeway et al. Immunobiology. The Immune System in Health and Disease Appendix 1.

The antibody used in the composition of the present invention may be directed to one of the following CD antigens and other cell surface markers: CDIa, CDIb, CDIc, CDId, CDIe, CD2, CD2R, CD3g, CD3d, CD3e, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CDlO, CDlIa, CDl Ib ₅ CDlIc, CDwl2, CD13, CD14, CD15, CD15S, CD15u, CD16a, CDlόb, CDwl7, CDl 8, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD44R, CD45, CD45RA, CD45RB, CD45RO, CD46, CD47, CD47R, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66a, CD66b, CD66c,

CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75,

CD75s, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CDlOO, CDlOl, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CDIlO, CDlIl, CDl 12, CDl 14, CDl 15, CDl 16, CDl 17, CDwI 19, CD120a, CD120b, CD121a, CDwl21b, CD122, CDwl23, CD124, CDwl25, CD126, CD127, CDwl28, CD130, CDwl31, CD132, CD133, CD134, CD135, CDwl36, CDwl37, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CDwl45, CD146, CD147, CD148, CD150, CD151, CD152, CD153, CD154, CD155, CD156a, CD156b, CD157, CD158a, CD158b, CD159a, CD160, CD161, CD162, CD162R, CD163, CD164, CD165, CD166, CD167a, CD168, CD169, CD170, CD171, CD172a, CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179a, CD179b, CD180, CD183, CD184, CD195, CD197, CD200, CD201, CD202b, CD203c, CD204, CD205, CD206, CD207, CD208 CD209, CDw210, CD212, CD213al, CD213a2, CDw217, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235ab

CD235b, CD236, CD236R, CD238, CD239, CD240CE, CD240D, CD241, CD242, CD243, CD244, CD245, CD246, CD247, 4-1BB Ligand

DR3, DR4, DR5, DR6, DcRl, DcR2, DcR3, HLA-ABC, HLA-DR, ICOS, ICOSL, OX-40, OX-40L, PDl, PD-Ll, PD-L2, TCRab, TCRgd, OPG, RP105, TNFRI, TRAIL, TWEAK, TWEAK Receptor, TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLRlO

Information (such as Other names, MW (kD), Structure, Cellular expression, Function and Gene) for these markers is available in the table at http://www.ebioscience.com/ebioscience/whatsnew/humancdchart .htm.

In particular, antibodies may be directed against: CD2, CD3, Cyto CD3, CD4, CD5, CD7, CD8, CDlO, CDlIb, CDlIc, CD13, CD14, CD15, CD16, CD19, CD20, Cyto CD22, CD22, CD23, CD25, CD33, CD34, CD36, CD38, CD41, CD42, CD45, CD56, CD57, CD61, CD69, CD79a, CD79b, CD103, CDl 17, CD138, TdT, HLADr, Myeloperox, glycophorin A or FMC7.

More in particular, the antibody may be directed to CD3, CD4, CD5, CD8, CDlO,

CD13, CD14, CD16, CD19, CD33, CD34, CD38, CD45, CD45RA, CD45R0, CD56 or CD57

The antibodies may also be directed against cytokines (such as IL-2, Merferon-gamma or tumour necrosis factor alpha) that are captured at the cell surface while being secreted by the cells that synthesize these moieties.

The most important single parameter in the monitoring of HTV infection is the expression of CD4 on cells. The absolute numbers of CD4+ lymphocytes and their percentage values within the total lymphocyte population (CD4+ T%/lymphocytes) are the most commonly requested flow cytometric assays. It is desirable to count T lymphocytes while avoiding the counting of CD4+ monocytes. For monitoring of viral infection (such as HIV infection), therefore, the composition may comprise a CD4 antibody.

The composition may comprise a combination of antibodies (such as two, three, four or more antibodies) such as: CD3, CD4, CD8, CD45; CD3,CD19,CD16,CD56; and CD4,CD8,CD38,CD45 or other similar combinations.

If the composition is to be used in the new "combined" method for flow cytometric HIV monitoring (see below under "Haematological tests"), it may comprise CD4, CD8, CD38 and optionally CD45.

The kit of the present invention would also be useful for diagnosing and monitoring leukemia cases in remote areas. A kit of two or three tubes with a panel of, for example, for or five antibodies per tube would allow samples to be taken directly for immunophenotyping and then transported to the referral centre. Again, the benefit would be stable sample conditions and the pre-staining of the sample for the subsequent assay.

CONJUGATION

If the antibody is for subsequent immumofluorescent analysis then it may be conjugated with one of the many fluorescent labels known in the art. For example, it may be labelled with FITC (fluorescein isothiocyanate), PE (phycoerythrin), Cy-5 (cyanin 5), PE-Cy5 (phycoerythrin-cyanin 5), APC (allophycocyanin), APC-Cy7, PE- Cy7, PC5 (phycoerythrin-cyanin 5.1), ECD (phycoerythrin-texas red), Alexa Fluor 488 or Alexa Fluor 647. If the composition comprises more than one antibody, then for two (or more) colour immunofluorescence each antibody needs to be conjugated to a different fluorochrome. An individual antibody may have one or more labels, making them one-, two-, or multi-coloured.

Antibodies suitable for use in the composition of the present invention are commercially available, for example Beckman Coulter™ produce ready-to-use conjugated monoclonal antibodies for flow cytometric analysis.

If the antibodies are to be detected by immunological detection methods other than those using immunofluoresence (such as by an ELISA) then they may be labelled with, for example, iodine, biotin or enzymes (such as horseradish peroxidase, alkaline phosphatase or β-Galactosidase) by methods commonly known in the art. Laballed antibodies are commercially available from firms such as Becton-Dickinson and Pharmingen, Caltag, Caltag-Medsystems, Dakomation, Chemicon.

Alternatively antibodies may be unlabelled, for example for indirect detection with a secondary reagent such as a labelled antiimmunoglobulin antibody or labelled Protein A.

CONTAINERS

In a second aspect, the present invention also provides a container which comprises a composition according to the first aspect of the invention.

The container may be suitable for receiving a cell sample, such as a sample of blood or bone marrow.

Preferably the container is suitable for receiving a cell sample, but does not yet comprise a cell sample.

The specimen collection container can, for example, be any suitable glass or plastics container of the type used in conventional blood collection systems for the collection of peripheral blood or bone marrow. The container can, for example, comprise a glass or plastic tube of capacity from 0.3ml to 10 ml, preferably about 5ml, excluding any air space, which has been rendered sterile by irradiation. Typically, the specimen collection container will have a volume capable of containing at least ten micro-litres, and preferably 20 to 200 micro-litres, for example, about 100 or 150 micro-litres of the composition of the first aspect of the invention. The container may, alternatively contain an equivalent volume in lyophilised form.

The container may also be in the form of a plate having a plurality of wells (such as 6, 12, 24, 48, 96 or more wells). Some or all of the wells may contain an aliquot of a composition according to the first aspect of the invention, in fluid or lyophilised form. The composition may vary between wells (for example with different antibodies or antibody combinations) or each well may contain an aliquot of the same composition.

In the collection method of the invention, blood or bone marrow may be drawn by any of the methods currently employed in the art of venesection directly into the specimen collection container. For individual tubes, aliquots of, for example, 900 or 1350ul of blood or bone marrow may be drawn into the specimen collection container of the invention which may contain, for example, 100 or 150 micro-litres of the composition of the first aspect of the invention, making a total volume of 1 or 1.5 ml respectively.

It is possible to perform flow cytometry with smaller volumes (such as 50μl) and this avoids wastage of reagents and reduces transport costs.

For small volumes (for example a 96-well microtitre plate) one option is to draw a volume (say 5ml) blood, then introduce aliquots (of perhaps 180ul) of this master cell sample to individual wells of the plate.

Smaller volumes, (particularly from a plurality of individuals) may also be obtained by finger prick (without excessive pressure on the finger) and transferred into the container by suitable micropipetting.

Obtaining blood from a fingertip is procedurally more simple and can be performed by a less skilled practitioner. In some parts of Africa there is a general aversion to needles and the sight of blood, making fingertip collection a more viable option than general venesection.

The ratio of the volume of the specimen to the volume of the composition is preferably

Preferably the container is suitable for use in a flow cytometer. Preferably the container is a tube which is adapted for direct transfer to the flow cytometer. Microplates with 96 wells may be directly counted on certain types of flow cytometers equipped with microplate readers such as the Luminex-100 (Luminexcorpration hie.) or the FACSArray (BDIS).

Flow cytometer tubes and 96-well microplates are widely available from firms such as Becton-Dickinson (such as TruCount™ tubes) and ELKAY.

The volume of the added blood may be known because it is ejected into the tube by a syringe at a known volume. Alternatively (or in addition) the container may be adapted to facilitate addition of a predetermined volume of cell sample. For example, the container may have a mark, such as a fill-line to show the level on the tube for the correct total volume of cell sample and composition. The mark may, for example be at a value between 0.5 and 1.5 ml, such as ImI.

The mark shows the level for the meniscus when the correct amount of blood is added to the composition already present in the tube, so blood is added until the line is reached.

Even when a known volume of cell sample is introduced to the container (for example by pipette or syringe), the mark would act as a double-check for the correct total volume.

The "mark" may be on the inside or outside of the tube and may be, for example, a line or a constriction in the internal diameter of the tube in a particular place.

The container may also have a "mark" to show the level on the tube for the correct amount of composition. This mark may, for example, be at 0.1ml.

The container may also be marked to show the volume within the container, either on a graduated scale (showing a plurality of total volumes such as 0.1, 0.2, 0.3, 0.4ml etc) or with just a few specific volumes (such as 0.5 and ImI).

Operating with known volumes is essential in some applications in order to carry out the calculation of the absolute numbers of cell types in the cell sample, and also to ensure that the active ingredients operate at the previously determined optimal concentrations. Alternatively, the samples collected (for exampple into containers containing lyophilized compositions) may be run on flow cytometers with absolute counting facility such as volumetry (e.g. Guava Easy Count system), bead assisted absolute counting (e.g. FACS Calibur with TrueCount beads and Beckman Coulter models with FlowCount beads) or instruments with a flow-rate calibration facility or any other absolute counting method.

Preferably the container is suitable for transportation. The container, or a plurality of containers may be adapted to be stably retained in a rack to maintain their orientation and stabilise tubes during transit and/or storage. The present invention also provides a rack comprising a plurality of containers according to the second aspect of the invention.

Tubes for use with TransFix™ and labelled by a meniscus to establish the volume of added blood are currently being designed (e.g. by Caltag Biosystems). There are companies that produce plastic tubes that contain the lyophilised fluorochrome conjugated antibodies (e.g. BIO.D, based in Barijtaly). The blood dissolves the

antibodies and the cells are stained. The present invention, however, includes both the fixative and the conjugated reagents. The advantage of this arrangement is the greater convenience and the higher precision of performing the assays.

KIT

In a third aspect, the present invention provides a kit which comprises a container according to the second aspect of the invention.

The kit may also comprise instructions for one or more of the following steps:

(i) how to use the container(s)

(ii) how to obtain the cell sample

(iii) how to transfer the cell sample to the container

(iv) mixing the sample with the composition

(v) storage/transport conditions (such as temperature etc)

(vi) analysis instructions

The kit may also comprise an explanation as to its purpose and the purpose of the or each different antibody (e.g. an explanation of the or each subset of cells intended to be stained by the or each different antibody).

The kit may also comprise cell sample collection apparatus (such as needles and/or syringes for blood samples).

Preferably the kit of the third aspect of the invention comprises a plurality of containers according to the second aspect of the invention.

The kit may comprise a plurality of containers each with the same antibody or antibodies. This may be for receiving aliquots of the same cell sample (perhaps for multiplication of results to ensure consistency; for example, equivalent results could be sent to different centres for analysis, perhaps for quality control purposes) or aliquots of different samples (such as blood samples from a plurality of individuals).

Alternatively (or in addition), the kit may comprise a plurality of containers each with a different antibody or mix of antibodies. For example, the kit may be designed to

receive an aliquot of the same cell sample to provide multi-layered information about this cell sample.

The or each tube in the kit may, for example, comprise one, two, three or four or more antibodies.

For the diagnosis and monitoring of HIV infection, it is useful to include a combination of antibodies such as:

CD4, CD45, CD8 and CD38 antibodies (see next section);

CD3, CD4, CD8, CD45;

CD3,CD19,CD16,CD56 or other similar combinations.

Preferably the kit includes either: a) a single-tube system wherein the or each tube comprises a mixture of the four antibodies (such as CD4, CD8, CD45 and CD38 antibodies); or b) a two-tube system which comprises

(i) a first container comprising two antibodies (such as CD45 and CD4); and

(ii) a second container comprising two antibodies (such as CD8 and CD38).

The kit may comprise a plurality of containers for the two-tube system to analyse a plurality of samples. This modified technique may be required if a relatively simple flow cytometric instrument is equippped with only two or three channels for detecting fluorescence.

HAEMATOLOGICAL TESTS

The present invention relates to a composition capable of fixing a cell sample (such as a blood sample) and staining it with one or more antibodies.

The invention is particularly applicable for the collection of blood or bone marrow for immunohaemotological analysis. Such analysis can be used in the investigation and diagnosis of, for example, acute and chronic leukemias, lymphomas,

immunodeficiencies, neuroblastoma, platelet disorders, and interstitial lung disease

(ILD).

The application of flow cytometry in the diagnosis and monitoring of hematologic malignancy is reviewed by Jennings and Foon (1997 Blood 90:2863-2892). This reveiw examines disorders such as Acute Leukemia (Acute Myeloid Leukemia, Acute Lymphoblastic Leukemia) Chronic Myelogenous Leukemia, Chronic Lymphocytic and pro lymphocytic leukemias, lymphoma, Mantle cell lymphomas, Follicular center cell lymphoma, marginal zone and related B-cell lymphomas, hairy cell leukemia, plasma cell dyscrasias, peripheral T cell disorders, Large cell Lymphoma, Hodgkin's disease, histiocytic neoplasms, aplastic anemia and myelodysplastic syndromes and lists the immunophenotypic markers characteristic for each condition. The composition of the present invention may be used in all or any of these analyses.

Four key applications for the technology of the present invention are (which are discussed in more detail below) are:

(i) the assay of CD34+ stem cells

(ii) the detection and monitoring of leukemia

(iii) the detection of Paroxysmal Nocturnal Hemoglobinuria (PNH) cells in blood

(iv) the assay of cytokines

(v) the preparation of samples for immunological and haematological monitoring for HIV infection.

CD34+ Cell Assay

The CD34 test has become the prominent test for measuring the stem and progenitor cell content of cell samples. The stem cells are distinguished from the majority of the cells in the bone marrow or blood because the stem cells display CD34 on their surface, which may be detected by flow cytometry. CD34 testing has the advantage (over, for example, the CFU test) that results can be available within 2 hours after receipt of the sample. This rapid turnaround time on CD34 testing can help prevent the patient from undergoing a needless second stem cell collection in cases where the first collection contained all the stem cells needed for the transplant.

The role of CD34 testing in the Oncology setting is to indicate when a certain predetermined level of CD34+ cells have been collected. This is important in minimizing the time a patient requires to begin the recovery from the transplant procedure. Many Oncology centers use certain goals that the patient must reach before they can be discharged. For example, the patient must reach specific levels of white blood cells (WBC) and platelets. As a result of the pre-transplant treatments, many patients' WBC and platelet levels will drop to dangerously low levels. The higher the transplant dose of CD34+ cells, the faster these WBC and platelet levels will return to normal levels.

hi addition to determining the adequacy of the stem cell collection for transplantation, the CD34 test can also be used to check on the patient's readiness to begin the stem cell apheresis collection. As a result of pre-collection treatment, CD34+ cells begin to circulate in the blood at high levels (termed mobilization). The level of circulating CD34+ cells in the blood is thought to be related to the amount of CD34+ cells that are eventually collected during the apheresis procedure. By starting the apheresis collections when the level of CD34+ cells has peaked in the blood, the number of collections that the patient must undergo is kept to a minimum.

Leukemia

For a review of the use of flow cytometry in the diagnosis and monitoring of leukaemia see Jennings and Foon (1997) Cancer invest 15(4) 382-399.

The technology of the present invention is particularly useful for diagnosis and monitoring of leukaemia in remote areas, where a kit of two or three tubes with, for example, four or five antibodies per tube could be used to fix and stain the samples where they are collected, ready for transportation to the referral centre.

Paroxysmal Nocturnal Hemoglobinuria (PNH) detection

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired, life-threatening disease of the blood. The disease is characterized by destruction of red blood cells

(hemolytic anemia), blood clots (thrombosis), impaired bone marrow function, and a 3 to 5% risk of developing leukaemia. PNH affects only 1-2 persons per million of the population and is a disease of young adults (median age of diagnosis 35-40 years of age) with occasional cases diagnosed in childhood or adolescence. PNH is closely related to aplastic anemia. In fact, up to 30% of newly diagnosed cases of PNH evolve from aplastic anemia. Similarly, the risk developing PNH after treatment for aplastic anemia with immunosuppressive therapy (anti-thymocyte globulin and cyclosporine) is approximately 20 to 30%. The median survival after diagnosis is 10 years; however, some patients can survive for decades with only minor symptoms.

Due to the wide spectrum of symptoms associated with PNH, it is not unusual for months or years to pass before the correct diagnosis is established. Some of the prominent symptoms of PNH include severe abdominal pain crises, severe headaches, back pain, excessive weakness, fatigue, and recurrent infections. The classic symptom of bright red blood in the urine (hemoglobinuria) occurs in 50% or less of patients. Frequently patients notice their urine is a dark tea-color. Typically, hemoglobinuria will be most noticeable in the morning, and clear as the day progresses. Attacks of hemoglobinuria may be precipitated by infections, alcohol, exercise, stress or certain medications. Many patients note a feeling of fatigue that may be disabling during periods of hemoglobinuria. The excessive fatigue does not appear to be related to the degree of anemia, as it improves when the hemoglobinuria abates. Blood clots (thrombosis) occur almost exclusively in veins, as opposed to arteries, and are the leading cause of death in PNH. Hepatic vein thrombosis (also referred to as Budd- Chiari syndrome) and sagittal vein (a vein in the head) thrombosis are the most common sites of thrombosis; however, all veins, especially those in the abdomen are susceptible.

A variety of blood tests are available for diagnosis of PNH. The sucrose hemolysis (sugar water) test and Ham test are available at almost all institutions, but can be falsely negative if the patient has received recent red blood cell transfusions. Over the past several years flow cytometry has become the gold standard for making the diagnosis. Flow cytometry has the advantage that the result is not affected by blood transfusions.

The assay of cytokines

Intracellular cytokines may also be ^" analysed by flow cytometry. Anti-cytokine antibodies are widely commercially available. It may be necessary to include a further reagent (such as a detergent) to fully permeabilise the cells. It may also be necessary to stimulate the cells in vitro in order to detect the cytokine by flow cytometry. Stimulation of cells with the appropriate reagent will depend on the cell type and the experimental conditions. For example, to stimulate T cells to produce IFN-gamma, TNF- alpha, IL-2, and IL-4, a combination of PMA (a PKC activator) and Ionomycin (a calcium ionophore) or anti-CD3 antibodies may be used. To induce IL-6, IL-10 or TNF-alpha production by monocytes, stimulation of PBMCs with LPS may be used.

It is also possible to combine the two procedures and analyse one or more cell surface molecules together with one or more cytokines.

HIV detection and monitoring

The progressive depletion of CD4+ T lymphocytes is the cardinal event in the pathogenesis during HTV infection. The absolute number of these CD4+ T cells in the peripheral blood is widely considered to be the most important parameter for monitoring the disease associated with HIV infection (Yeni et al (2002) JAMA 288:222-235).

For HIV diagnosis and monitoring, therefore, the composition of the present invention may comprise a CD4 antibody.

In Western countries, flow cytometric assays for CD4+ T cell enumeration are commonly designed for full lymphocyte subset analysis and include extensive quality controls. Some tests are CD3+ T-cell dependent, others CD45+ dependent, and some use both CD3 and CD45. Up to six different tubes may be included, each with a combination of two to four fluorochrome-labelled monoclonal antibodies (MAbs) (Brando, B., et al. Cytometry (Communication in Clinical Cytometry) 42, 327-346, Post et al (1996) QJM 89:505-508).

Simple "minimal" systems have been developed which are suitable for use in resource- poor countries. "Primary CD4 gating" (Sherman et al (1999) 222: 209-217; Janossy et al (2000) Br J Haematol 2000; 1198-208) and "primary CD45 gating" (Mandy et al (2003) MMWR Recomm Rep 52:1-13, based on work by Beverley et al. (1980) Nature 287: 332-3; Loken et al. (1990) Cytometry 11: 453-461. and also referred to as "panleucogating" (Glencross et al (2002) Cytometry 50:69-77) have previously been described.

Primary gating may involve just one antibody (e.g. CD4). Cell types are discriminated on the basis of their CD4+ expression and side scatter, enabling gating for the CD4+ T lymphocytes (CD4++, scatter +). This can be performed on both single and double platform machines (see below under "flow cytometry" and Janossy et al (2002 Clin Diag Lab Immunol 9: 1085-94+) and Janossy et al (2000) above). In a test of 552 samples, it has been shown that primary gating with a single CD4 monoclonal antibody (MAb) yielded absolute counts that were practically identical to those generated by a three-tube nine MAb protocol (Janossy et al (2000) as above). These simple tests are optimally performed on volumetric single platform flow cytometers. In these simple assays the discrimination between CD4+ T cells and CD4+ monocytes is fully maintained as opposed to other non-flow methods where monocytes can also erroneously contribute to CD4 counts (as discussed by Janossy (2004 Lancet 363: 1074)).

It was also shown that simple CD4 tests on volumetric flow cytometers could also generate CD4% values among the lymphocyte populations when the lymphoid cells were identified by their scatter morphology (Mercolino et al. (1996) Cytometry 22: 48- 59; Janossy et al. (2000) as above). Nevertheless, these assays can be more conveniently performed using two antibodies, CD45 and CD4. CD45, when used in primary CD45 gating, identifies all leucocytes (Beverley et al. (1980) Nature 287: 332- 3). Further, CD45 also discriminates between the different types of leucocytes, lympho-, mono- and granulocytes by their different staining intensity - to provide differential counts (Loken et al. (1990) Cytometry 11: 453-461). Thus CD45 antibody provides the white blood cell count (WBC) and differentials, and CD4 antibody provides the CD4 cell count, which in turn can be expressed as a proportion of the

WBC population (i.e. as CD4% among leucocytes and lymphocytes). This arrangement, especially when used on double haematological plus immunological platform, is referred to as panleucogating by Glencross et al. ((2002) Cytometry 50: 69-77).

This panleucogating protocol is most needed on double platform i.e. when the flow cytometer is not equipped to give absolute counts and the WBC absolute counts are obtained from haematology (Glencross et al. (2002) as above). Information on these methods for CD4+ cell enumeration is also available on the website http://www. AffordCD4.com.

These minimal systems have been the basis for a successful practical programme for resource-poor countries (Glencross et al (2002) Cytometry (Clin. Cytometry) 50:69- 77; Janossy (2002) Clin Diagn Lab Immunol 9:1085-1094; Janossy (2003) Clinical Applied Immunol Rev 4:91-107).

Although the CD4+ T cell count (together with the white cell differentials) is considered to be the central laboratory parameter in monitoring HTV patients, other information is important, in particular

(i) CD8+ cell counts

(ii) Total T cell counts (CD4 and CD 8 cells)

(iii) CD4/CD8 ratios

(iv) the proportion of CD8+ cells that are activated; and

(v) the HIV viral load.

A new, unified test has also been developed that provides information on T cell subsets and correlates with viral load, which is inexpensive, readily adapted to the new smaller, simpler flow cytometric machines and which utilises robust biological control procedures for quality assurance and gating.

The test involves the use of CD4, CD8 and CD38 antibodies. Advantageously a CD45 is also used to obtain white blood cell counts and differentials.

Enumeration of the CD4+, CD8+ and CD38+ populations within the cell sample, can provide information on the following:

(i) the CD4+ T cell counts

(ii) the CD8+ T cell counts

(iii) the total number of T cells (CD4 cell plus CD8 cell counts)

(iv) the CD4/CD8 ratio

(v) the number of highly activated (CD38++) CD8+ T cells, which is a surrogate marker for viral load

(vi) the number of CD38++ CD4+ T cells which appear in more advances HIV infection

(vii) the WBC counts and differentials.

The composition of the present invention, may for example, be used in the new unified test.

For example, a kit may be provided for use in a "two tube" test, which involves:

(i) obtaining a first and second aliquot of the cell sample;

(ii) treating the first aliquot with CD4 (and optionally CD45) antibodi(es);

(iii) treating the second aliquot with CD8 and CD38 antibodies.

The first and second treated aliquots may be analysed, for example, by two-colour immunofluorescence (IF).

It has been found that CD4+ and CD38+ monocyte populations show the same absolute monocyte counts. Hence, the CD4+ monocyte population within the first treated aliquot (or monocytes identified by both CD4 and CD45 criteria) and the CD38+ monocyte population within the second treated aliquot can thus be conveniently used to standardise the analysis between the two aliquots.

Alternatively, a kit may be provided for use in a "one-tube test". Here, the cell sample is analysed for its expression of CD4, CD8, CD38 and optionally CD45 simultaneously, for example using three- (CD4, CD8, CD38) or four colour immunofluorescence (CD4, CD8, CD38, CD45). This system has the advantage that all results are directly comparable (for example to obtain CD4/CD8 ratios).

The number of highly activated (CD38++) CD8+ T cells is a surrogate marker for viral load. Preferably the gate to separate CD8+ T cells into normal CD38+ cells and pathogenic CD38++ cells is set based on the CD38 expression of the monocyte population in a reference sample (for example, of normal or stabilised blood). Preferably the gate is set based on the mean, median or mode CD38 expression of the CD38+ monocyte population.

FLOW CYTOMETRY

The present invention relates to a composition for fixing and staining a cell sample. The sample may then be ready for analysis, for example using a flow cytometric method.

Flow cytometry is technique for recognising different cell types in cell suspensions and counting their subpopulations. A stream of cells, platelets or other microscopic particulate elements are passed through a beam of laser light. These events can be categorised by size (giving "forward scatter" FSC) and granularity (providing orthogonal "side scatter" SSC). Cells can also be tested with a variety of fluorochrome labelled antibodies that bind, most frequently, to cell surface moieties referred to as membrane markers. A laser based system is used to excite fluorescence. When hit by laser light the fluorochrome labelled markers emit signals at different wavelength. These signal combinations are detected and analysed by multi-parameter analysis. Cell sorting is usually not part of the analysis because even heterogeneous populations can be properly characterised by combinations of markers.

These studies generate a wealth of information using different fluorochromes on instruments equipped with multiple lasers. The great strength of the system is threefold. First, flow cytometers rapidly look at large numbers of individual cells and identify distinct cell populations with particular properties. Computer assisted analysis, mostly using uniform formats referred to FCS-files, enables determination of relative percentages of each specific cellular subset with great speed. Second, flow cytometric analysis is quantitative, both in terms of counting cell numbers and by documenting differences in the expression of molecules. Populations that express the same molecules in different concentration (e.g. CD4++ T cells displaying 5OxIO ³ CD4

molecules and CD4+ monocytes displaying 12xlO ³ CD4 molecules; Figure 1) can be readily resolved. This area of flow cytometry is referred to as Quantitative Flow Cytometry (QFCM). Non-flow methods designed to count cells frequently run into difficulty on these two criteria, the speed and quantitation, when compared to the performance of even the simplest flow cytometers. Thirdly, these instruments may not need to be complex and efficiently operate as diagnostically useful tool with a small laser that resolves two (or maximum three) fluorescence channels (Janossy et al. (2002) Cytometry 50: 78-85).

There is currently a drive to develop smaller and simpler flow cytometers particularly suitable for use in the developing world, hi a meeting in Bethesda 2002, Howard Shapiro MD advocated using inexpensive light-emitting diodes, plastics and digital technology to create dedicated diagnostic instruments that are easier to use than the complex flow cytometers designed for use in the research laboratories (reviewed on www.affordCD4.com).

The different arrangements to count absolute numbers of cells in blood or in a given cell suspension are reviewed on the AffordCD4 website as follows:

"Double platform" systems (reviewed in Brando, B., et al. Cytometry (Communication in Clinical Cytometry) 42, 327-346) employ two well-harmonized machines, a haematological counter plus a flow cytometer. These run parallel tubes of the same whole blood sample with no-wash technology. One of these tubes, analysed on the flow cytometer, is prestained with monoclonal antibodies. The crucial issue is, as explained below, that the two machines need to count a common parameter to precisely correlate the results obtained.

"Single Platform" systems are machines/platforms especially designed to count the absolute numbers of antibody labelled cells, equipped with multiple sample loader, programming facility and computer support. These are of two major types: 1. Volumetric flow cytometers count absolute numbers of cells in a given volume (eg ORTHO Cytoron™ and DAKO Galaxy™ and Guava.

2. Bead based systems that have no direct counting/ volumetric facility include (eg Trucount/ Flow Count™ counted on FACS Calibur™ or Coulter XL™).

In the "Varied problems -.varied solutions" section, a number of possible systems are discussed, including the following:

1. Volumetric flow cytometer using CD4 primary gating. This option has the highest throughput capacity, up to 400 samples per day, due to the possibility of using fully automated Windows-based software programmes with auto-bio-samplers of high volume. The combination of volumetry and a two-colour protocol with generic reagents on a single-platform contribute to an exceptionally high accuracy at a very low cost. The equipment, Cytoron-absolute made originally by Omron for ORTHO Diagnostics, is no longer available as new from the manufacturers but is still in use at specialised centres. This technology represents the optimal arrangement for regional centres defining the optimal specifications that the newly designed flow cytometers will need to meet.

2. The combination of any flow cytometer with any haematology analyser as a double platform using the recently introduced PanLeucogating protocol. This option allows for a relatively high throughput of samples with affordable cost. Only two reagents are needed, namely, CD45 and CD4 used in a two-colour combination. The additional CD45/CD8 double staining in a second tube is an optional extra. Costs can be further reduced by using generic reagents. The accuracy of absolute CD4 counts is superior to the protocols previously used on double platforms because the CD45 gating used here is more robust than the so-called 'scatter' -gating. Thus samples can travel for longer periods before the quality of immuno-phenotyping deteriorates. Clearly, this double- platform arrangement is well suited to haematology laboratories where Quality Assurance schemes are in operation for absolute white blood cell counts on the haematology analysers. Similar observations can be obtained by other newly developed systems such as the Guava, Inc. EasyCount™ system.

3. Any flow cytometers (from BDIS or Coulter-Beckman) with microspheres (beads) to assist single platform operation. This is the arrangement that is most fervently propagated as the optimal system for absolute CD4 T cell enumeration in Western

laboratories. Nevertheless, even in the West, the majority of laboratories still use double platform. This single platform arrangement delivers precise CD4 counts but at the expense of a relatively large reagent panel that includes expensive microbeads. Resource-poor laboratories that buy or get flow cytometers (perhaps even at a drastically reduced costs) loaded with an expensive reagent contracts are likely to find this system prohibitively expensive. Further, the clinicians are frequently not very pleased about the increased price wasted on a so-called 'full lymphocyte subset phenotyping service' when they require only affordable absolute CD4 counts, or, at most, CD4+CD8 counts. Hence the attractiveness of simplifying the staining protocols by utilising fewer reagents (e.g. CD45 plus CD4) during the routine service work even if it relies on a bead-based single platform operation.

4. FACSCount with its specialised reagent supply. A simple, robust version of dedicated flow cytometers, the FACSCount, has remained relatively expensive to run due to the high costs of the specialised reagents needed for its operation. The FACSCount can only process eight samples at a time as a suitable alternative for laboratories with limited sample load.

5. Red diode laser operated flow cytometers. Currently, two commercially available flow cytometers fall under this category, namely the CyFIo w and the LuminexlOO. The first one is designed for counting cells in clinical laboratories, while the latter one is designed for performing suspension arrays. Importantly, the current position is that both cytometers will need to undergo independent multi-centre validation trials before can be recommended for use in the routine clinical laboratory. The predictable arrival of newly designed, modern flow cytometers with inexpensive light sources will greatly contribute for the introduction of affordable CD4 testing, especially if such instruments operate reliable volumetric absolute counting with double colour immunofluorescence. Interestingly, the first experimental prototypes have proved the concept of red-diode laser instruments, and laser light sources emitting other then red colour are also constantly decreasing in price.

The fix and stained cell sample may be flow cytometrically analysed on either a single-platform or a double-platform system.

In its simple form, flow cytometry may use one, two, three or four immunofluorescence channels while analytical flow cytometry during research applications may utilise as many as 11-12 colours. Many fluorochrome labels are known in the art and include phycoerythrin (PE), fluorescein-isothiocyanate (FITC) and other fluorochromes selected on the basis of the instrument's light source and optical filter system. The protocols designed for resource-poor setting should remain simple to able to operate, preferably in one- or two-colour combinations.

CELL SAMPLE AKD SUBPOPULATIONS

The cell sample may be any sample that is a heterogeneous mixture of cells distinguishable by differences in their expression of cell surface markers. Preferably the cell sample is obtainable, directly or indirectly from a human or animal subject. For example, the cell sample may be (or be derivable from) a blood sample, a bone marrow sample, a body fluid sample, a disaggregated tissue sample or a tissue fine needle aspiration sample.

Preferably the cell sample is a blood sample. Blood samples may be obtained by methods known in the art such as aspiration using a syringe.

Normal blood comprises red blood cells, white blood cells and platelets. The white blood cell fraction comprises lymphocytes, granulocytes and monocytes basophils and eosinophils. The lymphocyte fraction, in turn, comprises T cells (such as CD4 and CD8 T cells), B and NK cells.

Of the total cells in the cell sample, different populations may be defined on the basis of their morphological characteristics and/or expression of cell surface markers (e.g. CD4+ and CD8+ cells). Within each population there may be one or more identifiable "subpopulations" (e.g. CD8+ cells which express CD38++).

METHODS

The present invention provide a method for preparing a cell sample for analysis which comprises the step of mixing the cell sample with a composition of the first aspect of the invention.

The mixing step may involve placing the cell sample into a container according to the second aspect of the invention, especially if the container is a specimen collection container.

The mixing step may also involve gentle shaking to mix the contents, perhaps by repeated inversion of the tube. Once mixed, the cell sample may be stored, either at room temperature (20-25 ⁰ C) or in a chilled environment (about 4°C). According to Jani (2001, above) when whole blood is fixed with TransFixTM, absolute CD4+ counts remain stable for at least 10 days when stored at 20-25 °C and for 7 days when stored at 37 ⁰ C, to mimic tropical conditions.

The cell sample may also be mixed with an anticoagulant either before, during or after mixing with the composition of the present invention.

DISEASES

The disease may be any disease which causes a change in the properties of a cell sample from the patient which is identifiable by staining with an antibody.

In particular, the disease may be one which causes alterations in the level of expression of a cell surface marker, or an intracellular marker (such as a cytokine) on a population of cells in a cell sample.

The disease may be associated with or treatable by transplantation of CD34+ stem cells.

The disease may be Paroxysmal Nocturnal Hemoglobinuria (PNH).

The disease may be associated with an alteration in the levels of one or more cytokines by a population of cells in a body fluid or tissue.

The disease may be or cause an immune deficiency. The composition of the present invention is contemplated for use in a method for identifying and monitoring diseases which affect the number of CD4 and/or CD8+ cells of an individual. Immune deficiencies may be related to a decrease in the number of these cells and/or to the alteration of the relative ratio of the cell populations.

The disease may be a viral infection. Viral infection causes the number of activated CD8+ T cells to increase, which can be detected as in increase in CD8+/CD38++ cells. These excess cells carry mostly the CD45R0+,CD45RA-negative phenotype.

The composition may be used to identify and/or monitor HTV infected individuals. As HIV infected individuals progress towards AIDS, their CD4+ T cell count decreases from normal levels (>800 CD4+ T cells/ul blood) to less than 200 cell/ul (Phillips et al (1991) Lancet 337: 389-392).

The levels of CD8+/CD38++ cells increases during a viral infection. For most viral infections this will be a transient increase, but in HIV infected patients (if untreated) the number of these pathogenic T cells progressively increases. Both investigations in the simian models and long-term follow-up in patients indicate that lymphocyte activation shows more direct relationship to the pathology then viraemia itself (reviewed by Iyengar et al. (2003) Lancet 362: 945-950). Then at the late stages of HIV disease CD8 lymphopenia develops and the residual CD4+ T cells increase their CD38 expression (Giorgi et al. (1999) as above). The method can therefore be used for HIV infected patients to monitor the underlying rise in these activated T cells as well as any transient increases which may be due to opportunistic infections.

Antiretroviral therapy (ART) is becoming available to poorer areas of the globe. For ART to be used most effectively, it needs to be administered to the right patients at the right time and in the right amount. The therapy itself needs to be monitored so that it can be continually adjusted.

The composition of the present invention may be used in a method: (i) to diagnose a disease

(ii) to monitor the progression of the disease in an untreated patient (iii) to optimise the timing and/or nature of treatment program (iv) to monitor the patient during treatment, for example to gauge efficacy . (v) to investigate the relationship between the recovery of CD4 T cell population and CD8 activation and HIV load while patients are on ART, and/or

(vi) for disease surveillance, e.g. to estimate the need for health care in a particular region.

(vii) to assist the monitoring of opportunistic infections during HIV infection, with a view to optimising an appropriate treatment regime.

Since TransFix™ may stabilise cell samples for more than 10 days (Jani et al (2001) as above), if a sample is fixed and stained using the composition of the present invention it may be analysed at a time and/or location distant from that of its collection. In the Jani paper, samples were fixed and transferred between continents for analysis seven days later. This facilitates sample collection in the field in developing countries followed by transport to a second site for analysis. The assays may be performed at local/regional laboratories (for example using the inexpensive Cd4+ counting method) or transferred to more sophisticated analysis equipment overseas. The length of stability of the samples also enables intercontinental transport of samples. This may be important, for example, to enable quality control measures of performance to be taken involving internationally approved Quality Assurance Centres.

The sample may be analysed, for example, between 1 and 30 days after it is fixed and stained. Preferably the sample is analysed 10 or fewer days, for example between 3 and 7 days, after it is fixed and stained.

The sample may be transferred to a different site within the same country, to a different country within the same continent, or to a different continent before analysis.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLES

Example 1

To investigate the capacity for a pre-mixed composition of TransFixTM and antibody to fix and stain a cell sample, 900ul of normal blood was added to tubes containing lOOul of a mixture of TransFix™ and CD4 antibody that has been labelled with Alexa- 488-dye at various dilutions (and anticoagulants). The sample is gently mixed and retained at room temperature for varying length of time (Fig.l).

Figure 1 shows the staining by saturating amounts of antibody (5ug/ml-Fig 1 A), and its 100-fold (Fig IB and E) and 200-fold (Fig 1 C and F) dilution. The same sample has also been stained with the same antibody dilutions, kept in the presence of Transfix for 14 days (Fig 1 E and F). Fig ID shows the negative control.

It is shown that there is only a minimal loss of staining intensity due to this storage, from MFI value 90 to 80 at the 100-fold dilution and from MFI value 45 to 30 at the 200-fold dilution. At the same time the CD4+ T lymphocytes are readily counted in the suspension without an alteration of the percentage or absolute counts (31.4% of lymphocytes throughout the tests). It can be concluded that the Transfix is not denaturing the CD4-antibody during a 2-week storage period when the tubes are kept in at 4CO. Similar observations are seen when the test is performed using CD4 antibodies labelled with fluorescein-isothiocyanate (FITC), data not shown.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the

invention as claimed should not be unduly limited to such specific embodiments.

Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.