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Title:
KIT FOR IN PROCESS QUALITY CONTROL TO PREDICT AND DETERMINE ENGRAFTMENT AND MULTILINEAGE RECONSTITUTION POTENTIAL OF HEMATOPOIETIC CELLS AND CORD BLOOD STORAGE
Document Type and Number:
WIPO Patent Application WO/2006/052852
Kind Code:
A2
Abstract:
The present invention relates generally to kits that provide reagent mixes and instructions for the use thereof, in performing high-throughput assay methods for stem cell transplantation and cord blood storage quality control and methods of use. The methods measure the luminescent output derived from the intracellular ATP content of incubated target cells, and correlate the luminescence with the proliferative status of the cells. By non-subjectively detecting and quantitatively measuring the proliferation potential of several hematopoietic stem and progenitor cell populations simultaneously in a prospective or even retrospective manner, the present invention can determine the "quality", engraftment, repopulation and multilineage reconstitution potential of the infused cells, thereby significantly increasing the ability to predict the transplant outcome and reducing risk to the patient.

Inventors:
RICH, Ivan, N. (530 Paisley Drive, Colorado Springs, CO, 80906, US)
Application Number:
US2005/040222
Publication Date:
May 18, 2006
Filing Date:
November 04, 2005
Export Citation:
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Assignee:
HEMOGENIX, INC. (Suite D, 4405 North Chestnut StreetColorado Springs, CO, 80907, US)
RICH, Ivan, N. (530 Paisley Drive, Colorado Springs, CO, 80906, US)
International Classes:
C12Q1/00
Domestic Patent References:
2003-01-16
Foreign References:
US20020146680A12002-10-10
Other References:
KIESSLICH, T. ET AL.: 'Fast abd Reliable Determination of Intrascellulasr ATP from Cells Cultured in 96-Well Microplates' JOURNAL OFBIOCHEMICAL AND BIOPHYSICAL METHODS vol. 57, no. ISSUE, 2003, pages 247 - 251
Attorney, Agent or Firm:
ROSEMBAUM, David G. (Rosembaum & Associates, P.C.650 Dundee Road,Suite #38, Northbrook Illinois, 60062, US)
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Claims:

WHAT IS CLAIMED IS;

1. A method for determine engraftment and multilineage reconstitution potential comprising: (a) preparing one or more cell suspensions, (b) adding the one or more cell suspensions to a pre-dispensed and pre-mixed tube comprising a serum mix, a methyl cellulose mix, a growth factor mix, a medium, an ATP-releasing reagent, and an ATP luminescence-monitoring reagent thereby forming one or more samples, (c) dispensing one or more samples into one or more vessels, (d) incubating for 7 days at 37 C in a humidified atmosphere with 5% CO 2 and 5% O 2 , (e) performing an ATP standard dose response and measuring luminescence, (f) measuring relative luminescence of the one or more samples, (g) conversion of relative luminescence units of the one or more samples to standardized ATP units and (h) correlating the standardized ATP units to engraftment and multilineage reconstitution potential.

2. The method of claim 1 wherein the cell suspension comprises colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), high proliferative potential stem and progenitor cell (HPP-SP), burst-forming unit erythroid (BFU-E), granulocyte-macrophage colony-forming cell (GM-CFC), megakaryocyte colony-forming cell (MIc-CFC), T cell colony-forming cell (T-CFC), B cell colony-forming cell (B-CFC), colony-forming cell-blast (CFC-blast), high proliferative potential colony forming cell (HPP- CFC), macrophage colony-forming cell (M-CFC), granulocyte colony forming cell (G-CFC), colony-forming unit-erythroid (CFU-E), colony-forming cell-basophil (CFC-Bas) or colony- forming cell-eosinophil (CFC-Eo) or any combination thereof.

3. The method of claim 3 wherein one or more growth factors are added to the one or more samples.

4. The method of claim 3 wherein the cell suspension comprises B-CFC and the growth factor comprises IL-7.

5. The method of claim 3 wherein the method further comprises adding a background control that does not contain the one or more growth factors.

6. The method of claim 1 wherein the cell suspension is derived from normal blood, peripheral blood, bone marrow or umbilical cord blood.

7. The method of claim 1 wherein the one or more cell suspensions is thawed from one or more frozen cell suspensions.

8. The method of claim 1 wherein the one or more cell suspensions comprises one or more stem cell subsets.

9. The method of claim 8 wherein the one or more stem cell subsets is selected from the group consisting of CD34, CD 117, CD 133 and CD90.

10. The method of claim 1 wherein the one or more cell suspensions has a concentration of about 0.5 x 10 6 cells/ml or about 0.5 x 10 4 cells/100 μl culture.

11. The method of claim 1 wherein the one or more cell suspensions is purified to CD34 + cells and has a concentration of about 1 x 10 4 cells/ml to about 2 x 10 4 cells/ml or about 1 x 10 2 cells/100 μl culture to about 2 x 10 2 cells/100 μl culture.

12. The method of claim 1 wherein incubating for 7 days at 37 C in a humidified atmosphere with 5% CO 2 and 5% O 2 , the one or more samples are transferred to a 23 C humidified incubator with 5% CO 2 and left to equilibrate at least 30 minutes.

13. The method of claim 1 wherein the serum mix comprises bovine serum albumin, an insulin, an iron-saturated transferrin, a serum and IMDM.

14. The method of claim 1 wherein the growth factor mix comprises at least one growth factor selected from the group consisting of erythropoietin, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2, interleukin- 3, interleukin-6, interleukin-7, interleukin-15, Flt3L, and leukemia inhibitory factor, and combinations thereof.

15. The method of claim 1 wherein the methyl cellulose mix has between about 1.5% and about 2.5% methyl cellulose.

16. The method of claim 1 wherein the concentration of fetal bovine serum in the cell growth medium is between about 0% to about 10% by volume.

17. The method of claim 1 wherein the concentration of methyl cellulose in the cell growth medium is about 0.7% by weight.

18. The method of claim 1 wherein the method further comprises contacting the target cell population with at least one cytokine.

19. The method of claim 18 wherein the at least one cytokine is selected from the group consisting of erythropoietin, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L, leukemia inhibitory factor, and combinations thereof.

20. The method of claim 1 wherein the reagent capable of generating luminescence in the presence of ATP comprises luciferin and luciferase.

Description:

TITLE OF THE INVENTION

KIT FOR IN PROCESS QUALITY CONTROL TO PREDICT AND DETERMINE ENGRAFTMENT AND MULTILINEAGE RECONSTITUTION POTENTIAL OF

HEMATOPOIETIC CELLS AND CORD BLOOD STORAGE

INCORPORATION BY REFERENCE

This application claims benefit of U.S. Provisional patent application Serial No. 60/624,993 filed November 4, 2004. Reference is also made to U.S. patent application Serial Nos. 10/059,521 filed January 29, 2002 and 10/645,077 filed August 21, 2002. The foregoing applications, and all documents cited therein or during their prosecution ("appln cited documents") and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. FIELD OF THE INVENTION

This application relates to the development and validation of a specially developed Hematopoietic/Hematotoxicity Assays via Luminescence Output ("HALO") kit for stem cell transplantation and cord blood storage quality control and methods of use. BACKGROUND OF THE INVENTION

Hematopoietic stem cell transplantation (HSCT) and, possibly in the future, embryonic stem cell transplantation, require assays to ensure that the processed cells to be transplanted into a patient will home to the designated target organ, engraft or seed in that organ, initiate proliferation and finally reconstitute or repopulate the organ or tissue.

Transplantation of the hematopoietic system can utilize stem cells present in the bone marrow (the primary site of adult hematopoiesis), peripheral blood which contains circulating stem cells, and umbilical cord blood, which contains fetal-derived stem cells and considered less mature and naive with respect to their development. Depending on the stem cell source, HSCT can be performed in an autologous manner in which the patient receives his or her own processed stem cells or in an allogeneic manner in which the patient receives stem cells from a different person. Regardless of the source and the type of transplantation performed, the quality of the stem cells transplanted must be ensured since the patients receiving the stem cell infusion have had their hematopoietic system partially or totally ablated by radiation and cytotoxic drugs and are at increased risk of dying if the transplanted cells do not engraft and

repopulate their hematopoietic system. Although the first human autologous bone marrow transplantation (BMT) was performed by Kurnick et al in 1958, no assays to determine the quality of human transplanted cells with respect to their growth and engraftment potential were available until Pike and Robinson in 1971 applied the in vitro colony forming assays first discovered in 1966 to human cells. With the introduction and use of flow cytometry and reagents to measure stem cell membrane antigens and viability beginning in the late 1980s, the use of the colony- forming assay (CFA) has declined and has almost been eliminated from HSCT procedures in the U.S. One of the major reasons for this is that it takes 14 days for the cells to grow in culture and obtain a result. This is about the same time as it takes to observe engraftment in a patient. Therefore, the CFA has been considered of little use or completely superfluous. True, flow cytometry can measure the viability and number of cells expressing the stem cell antigen CD34, and other lineage-specific antigens, but this provides no direct information as to whether the transplanted cells will exhibit growth potential in the patient, leading to engraftment and balanced multilineage reconstitution. Despite its disadvantages (of which there are unfortunately many), the colony-forming assay is the only assay available that can reliably measure repopulation potential of transplanted stem cells and the potential for multilineage reconstitution.

The term transplantation as defined by the Foundation for the Accreditation of Cell Therapy (FACT, previously called the Foundation for the Accreditation of Hematopoietic Cell Therapy, FAHCT) "is the infusion of autologous, syngeneic or allogeneic hematopoietic stem cells with the intent of providing transient or permanent engraftment in support of therapy of disease". The term hematopoietic progenitor cell has been used to include primitive pluripotential hematopoietic cells capable of self renewal as well as maturation into any of the hematopoietic lineages. Despite the fact that this term has been used for many years, the aim of this type of transplantation is to infuse both short- and long-term hematopoietic stem cells in order to replace and repopulate the hematopoietic system of a patient. Progenitor cells refer to the direct lineage-committed descendants of stem cells and therefore do not exhibit self-renewal capability and have little value to the patient who has been transplanted. Throughout this proposal, the term hematopoietic stem cell transplantation (HSCT) will be used in place of hematopoietic progenitor cell transplantation or therapy.

Stem cell transplantation is considered a form of cell therapy. Although many animal transplantation experiments were published prior to 1961, quantitative assessment of stem cell transplantation in a murine model was demonstrated by Till and McCulloch. The first

human autologous bone marrow transplant was performed in 1958 by Kurnick et al. This was followed a year later by Thomas et al who demonstrated that syngeneic bone marrow could rescue a patient with leukemia after total body irradiation and by McGovern et al who performed the first autologous bone marrow transplantation (BMT) for acute lymphocytic leukemia (ALL). Thomas and Storb described the first standardized technique for bone marrow harvesting in 1970. The use of various anti-proliferative drugs began with cyclophosphamide for the treatment of leukemia in 1975. Civin and coworkers identified the CD34 antigen in 1984 and in 1991, Berenson et al showed that autologous CD34 + cells could reconstitute hematopoiesis in patients receiving myeloablative therapy. The first gene- marking study was performed by Brenner and coworkers in 1993.

Following the detection and identification of early human hematopoietic progenitor cells in vitro by Pike and Robinson in 1970, a number of groups detected these cells in human PB in 1971. Richman et al demonstrated an increase in progenitor cells in PB following chemotherapy and the first autologous peripheral stem cell transplant (PSCT) for chronic myeloid leukemia was performed by Goldman et al in 1979. In 1988 Socinsksi and colleagues demonstrated that human recombinant granulocyte-macrophage colony- stimulating factor (rHuGM-CSF) expanded circulating blood cells. A year later, Duhrsen et al showed that granulocyte-colony-stimulating factor (rHuG-CSF) increased hematopoietic progenitor cells in the peripheral blood. This process is now called stem cell mobilization. Between 1987 and 1990, a number of reports showed mobilized blood cells could accelerate hematopoietic recovery compared with bone marrow, hi 1992, G-CSF was used to mobilize autologous PSCT and several reports demonstrated the use of G-CSF to mobilize syngeneic and allogeneic PSCT. In 1995, Brugger et al demonstrated that CD34 + selected cells expanded ex vivo could successfully enhance hematopoiesis after intensive therapy. Although placental blood had been used as a source of blood transfusion as early as

1939, it was not until 1974 that Knudtzon reported the presence of in vitro granulocyte- macrophage colony-forming cells (GM-CFC) in UCB using the technique reported by Pike and Robinson. The biological properties, presence of stem cells in UCB and its use in relation to transplantation was reported by Broxmeyer et al. However, the first UCB transplantation was performed by Gluckman and colleagues in 1989 and was followed by several other reports, which resulted in the first International Conference Workshop on Cord Blood Transplantation Biology and Immunology in 1993: Shortly thereafter, cord blood banking was started by the New York Blood Center with other companies such as the Cord Blood Registry and ViaCord/ViaCell providing commercial cord blood banking for parents

wanting to preserve cord blood stem cells for their children in the event of transplantation requirement in later life.

Regardless of whether bone marrow, peripheral blood or cord blood is used as a source of hematopoietic stem cells for transplantation, the cells have to be processed in order to separate the stem cells representing approximately 1-4% of the population from the bulk. Bone marrow is usually collected from donors under general anesthetic from the iliac crest. Approximately, 100ml or more marrow is taken. Processing of the bone marrow now involves sophisticated technology to separate the CD34 + cells fraction (containing stem cells and progenitor cells) and, at the same time, deplete the fraction of a certain proportion of T- cells that reduce the incidence of graft- versus-host disease (GvHD), a procedure that dates back to the early 1980s. Although aliquots are tested and some are frozen, in general the processed bone marrow is infused into the patient directly after it is processed. Peripheral blood contains a low, but relatively constant number of circulating stem cells. These can be greatly increased by administering growth factors such as GM-CSF and G-CSF to mobilize the stem cell pool from the bone marrow into the periphery in autologous recipients or allogeneic donors for family members receiving the transplant. The growth factor is injected over several days and on each day the recipient/donor is linked to an apheresis machine which is designed to optimize the collection of peripheral blood components containing the stem cells, namely the mononuclear cell (MNC) fraction, and remove mature blood cell components. Between 12-15 liters of blood are processed in this manner on various days after growth factor administration. Each collection contains about 87% MNCs and the cells are transferred to a bag and cryopreserved after an aliquot is removed for testing (see below). After several aphereses and preparation of the patient for transplantation, the cells are thawed, processed and infused back into the patient. Umbilical cord blood is collected from the placenta either while still in utero or after it has been delivered. The cord blood is collected by gravity into a bag and then processed. A cord blood unit can be 100ml or more. Red cell depletion and volume reduction now uses Hespan, a product composed of 6% hetastarch in 0.9% saline from DuPont Pharma. After removing sufficient cells for testing (see below), the cord blood cells are cryopreserved and stored in liquid nitrogen until needed. The cell processing laboratory (CPL) is responsible for the success of the transplant.

To this end, standards to maintain and enhance the quality and safety of the transplantation process through inspection and accreditation have been controlled by two groups in the United States, namely the American Association of Blood Banks (AABB) and FACT and in Europe by the Joint Accreditation Committee of ISCT-Europe and EBMT (JACIE). The U.S.

Food and Drug Administration (FDA) has' provided guidelines, especially since the implementation of gene therapy and ex vivo hematopoietic stem cell expansion protocols, and it appears that it will take some time for the FDA to produce regulations in this area due to the rapidly evolving field of stem cell research. For all tissue sources, quality control includes testing for sterility, contamination by microorganisms and viruses, HLA and blood type matching and, at each stage of the operation, standard operating procedures (SOP) document all the processes and procedures. However, as far as ensuring that sufficient numbers of viable stem cells with the necessary growth potential are concerned, standards from both the U.S. and Europe are distinctly lacking in information. There are 2 primary reasons for this. First, a lack of standardized, robust and non-subjective assays and second, a lack of consensus regarding the procedure(s) to be used. To illustrate this point, the JACIE standards state in Section D4.270, "For products undergoing manipulation that alters the final cell population, a relevant and validated assay, where available, should be employed for evaluation of the target cell population before and after the processing procedure(s)". In most instances, however, there are 4 parameters that are or have been used. These are nucleated cell count, viability and CD34 + cell number by flow cytometry and "progenitor cell assays" to monitor stem cell procedures and graft manipulations.

Nucleated cell count is an integral part of the cell processing procedure because at every step, the number of nucleated cells has to be determined in order to evaluate cell yields, recoveries and purity. This was originally performed using a hemocytometer, but instrumentation that not only evaluates the nucleated cell count, but also the different cellular components is now used in the CPL. The use and validation of these instruments is controlled by proficiency testing.

The viability of the product provides an indication as to the proportion of live or dead cells that are transplanted. This too was originally determined by dye exclusion using trypan blue and a hemocytometer. Nowadays, viability is performed in conjunction with the CD34 + count determined simultaneously by flow cytometry. Fluorochromes such as 7- aminoactinomycin D (7 -AAD) are reliable and easy to use indicators of viability.

Due to several problems inherent in the colony-forming assay (discussed below), and the discovery of the CD34 membrane antigen in 1984 by Civin et al., the use of flow cytometry instrumentation to detect hematopoietic cell populations and their subsets has increased 'dramatically. In 1992, the First European workshop on Peripheral Blood Stem Cell Determination and Standardization occurred. The participating investigators were called the Milan-Mulhouse Group. It was from this group that the first flow cytometric protocol for

the estimation of CD34 + cells was developed. This was the beginning of the use of flow cytometry as known today for measuring CD34 + cells and its use in diagnosing many different diseases, including malignant hematopoietic diseases. Since that time, detection and quantifying CD34 + cells and their subsets has been studied by numerous groups to standardize and validate the use of this procedure for stem cell transplantation purposes. This has recently been reviewed by Gratama and coworkers. The flow cytometric procedure used by most transplant centers is called the ISHAGE single platform or protocol, which allows enumeration of absolute CD34 + cell numbers. This protocol has reduced the subjectivity of the gating procedure, since depending how regions and gates are set in flow cytometry, different results can be obtained. Depending on the tissue source and manipulation, CD34 + cells represent between 1% and 4% of the total population. However, there are 3 classes of epitope that are recognized by the CD34 monoclonal antibody. The antibodies used for clinical purposes recognize class II and III. However, although generally considered the human stem cell antigen, the CD34 antigen is present not only on a subsets of 3 stem cells, but also virtually all lineage-determined progenitor cells, including those defined by the colony-forming assays as burst-forming units — erythroid (BFU-E), GM-CFC, megakaryocyte colony-forming cells (Mk-CFC) and even T- and B -colony-forming cells (CFC). Most of the lineage committed progenitor cells express the CD38 antigen. It has therefore been possible to distinguish between total CD34 + cells and CD34 + /CD38- stem cells. Of particular importance for the present discussion is the observation that correlation of the number of GM-CFC (also called CFC-GM, CFU-GM or CFU-C) with the number of CD34 + cells, has provided the validation to replace the CFA with flow cytometric analysis for quality control, engraftment and reconstitution. It should be emphasized that such a correlation between CFA and CD34 + cells does not always exist in which 173 paired assays of GM-CFC and CD34 + performed during 1996 and 2000 on transplanted cells, have been correlated. No correlation was found between the nucleated cell counts and either GM-CFC or CD34. However, over the last 10 years, the minimum CD34 + cell dose accepted to be safe for transplantation has actually decreased from about 10 x 10 6 /kg to about 2-2.5 x 10 6 /kg, although there is no general consensus, except that more is better and megadoses of CD34 + cells (>10 x 10 6 /kg), improves the rate and completeness of engraftment across major histocompatability barriers.

Besides the aforementioned possible subjectivity involved in flow cytometric analysis performed during cell processing and after transplantation, several other assumptions are being made. First, as mentioned above, the CD34 + cell population not only contains stem

cells exhibiting different degrees of "sternness" and therefore different repopulating capacities, but also progenitor cells that have no self-renewal capacity and very limited repopulation ability. Second, although viability of CD34 + (CD38-) cells is usually measured, this does not mean that the CD34 + stem cells have long- and/or short-term proliferation or growth potential. Third, measuring viability and assessing different subpopulations of transplanted cells is, like the CFA, a surrogate assay that in this case, measures membrane antigen expression that can be indicative of engraftment and multilineage growth, but does not directly detect or measure stem cell growth and multilineage reconstitution potential. Acceptance of these assumptions and the failure to measure growth potential of the transplanted cells can lead to graft failure and potential increased danger to the patient.

Prior to the introduction of flow cytometry, the colony-forming assay or "progenitor cell assays" as they have been called in the transplantation field, were and indeed still are, the only surrogate assays that can detect and measure (i) the presence of stem and progenitor cells during the cell processing procedure, (ii) predict the ability of the transplanted cells to engraft and (iii) predict and detect multilineage repopulation potential. The first in vitro colony- forming cell population to be detected was the GM-CFC in the mouse bone marrow. The human equivalent was detected by Pike and Robinson in 1970. As mentioned above, the GM-CFC is a bipotential committed, progenitor cell population, since when stimulated with GM-CSF, granulocyte, macrophage and mixed colonies are produced. This population was originally used for transplantation quality control purposes, and hence term progenitor cell assays and progenitor cell transplantation. Since the discovery of the GM-CFC population, in vitro progenitor and precursor populations for all lympho-hematopoietic lineages as well as several in vitro stem cell populations have been detected. Although it might seem obvious to continue to use the colony-forming assays to detect the growth potential of different stem cell populations or multiple lineage-specific progenitor cell populations present in the tissue transplanted into patients, today, only a handful of transplant centers still routinely perform the colony-forming assays for quality control purposes. The primary reasons for this have been discussed by Burt, Deeg, Lothian and Santos in 1996 in their manual entitled "On Call In .... Bone Marrow Transplantation". In their discussion of hematopoietic progenitor stem cells they state, "...graft evaluation on the basis of clonogenic (colony-forming unit or cell) assays had two major drawbacks for clinical use in transplantation: the assays took 2 weeks to complete, so that results were available only after transplantation; and 2) murine studies suggested that the majority of clonogenic cells were not responsible for long-term hematopoietic reconstitution." Although the first statement was is true, the second was not

since virtually everything known about short- and long-term reconstitution initially came from the murine transplantation model. Later in the same manual, in a section entitled Product Assessment/Quality Assurance, the colony-forming units are again discussed. "Until recently, the most commonly used assay for reconstituting ability of a graft was the growth of the 14 day granulocyte 4 macrophage colony-forming unit (CFU-GM) in semisolid medium. Threshold doses for CFU-GM differ widely, but may be in the range of about 0.1-1 x 10 4 /kg for marrow, and about 1-5 x 10 5 /kg for blood-derived stem cells. Unfortunately, culture conditions vary among laboratories, making comparisons difficult. Further, since the cultures cannot be counted until 14 days after a harvest, real-time evaluation of a graft is impossible." In an article by Henon et al in 2001 the authors state, "Determination of the graft content in CFU-GM was the only one available until the end of the eighties. But, for technical reasons, and also because it does not actually evaluate the self-renewal potential of the cell products reinfused, it has now been commonly replaced by the determination of CD34 + cell amounts, which are known to contain the pluripotent hematopoietic stem cells." The detection of human in vitro multipotential stem cells had been reported in the late 1970s. The clinical stem cell transplantation community continued to use the CFU-GM assay when an assay was available that could directly detect and measure multipotential stem cell engraftment. This CFC-GEMM (colony-forming cell granulocyte, erythroid, macrophage megakaryocyte) assay was readily available in the early 1980s and shown to be particularly useful to detect stem cells in different hematological diseases. Despite the fact that the success of hematopoietic transplantation is based on the low frequency of stem cells, this argument has been used against the CFC-GEMM and other stem cell populations as a means of quality control and assessment. However, like the CFU-GM, the CFC-GEMM population requires the same amount of time to grow and form colonies, namely 14 days. Unfortunately, there are several other drawbacks to the conventional colony- forming assay. The assays are time-consuming to perform, they require a high degree of technical expertise to manually enumerate the colonies, the assay is highly subjective and there is a lack of standardization in colony enumeration. In short, many aspects lead to a cumulative variation so that results within and between different laboratories are very difficult to compare. Despite these drawbacks, there are 3 reasons for keeping the basic assay procedure. First, they have been part of the research and clinical community since their inception; second, they have been proven and validated worldwide, and third, no other procedures, other than the colony-forming assays exists to determine the quality of growth potential of the stem and progenitor cells populations required for engraftment and multilineage reconstitution. It should also be mentioned that

besides the introduction of serum-free conditions and recombinant growth factors, the CFA procedure has changed little since 1966.

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. SUMMARY OF THE INVENTION

The hypothesis upon which this application is based in part, is that by non- subjectively detecting and quantitatively measuring the proliferation potential of several hematopoietic stem and progenitor cell populations simultaneously in a prospective or even retrospective manner, the 7 day assay can determine the "quality", engraftment, repopulation and multilineage reconstitution potential of the infused cells, thereby significantly increasing the ability to predict the transplant outcome and reducing risk to the patient.

The present invention relates generally to kits that provide reagent mixes and instructions for the use thereof, in performing high-throughput assay methods that predict and determine the engraftment and multilineage reconstitution potential of cell populations. The methods measure the luminescent output derived from the intracellular ATP content of incubated cells, and correlate the luminescence with the proliferative status of the cells. The kits and methods of the present invention address the need for rapid assays that will determine the proliferative status of hematopoietic stem and progenitor cells and of subpopulations of differentiated cells thereof. The invention relates to a method for determine engraftment and multilineage reconstitution potential which may comprise (a) preparing one or more cell suspensions, (b) adding the one or more cell suspensions to a pre-dispensed and pre-mixed tube comprising a serum mix, a methyl cellulose mix, a growth factor mix, a medium, an ATP-releasing reagent, and an ATP luminescence-monitoring reagent thereby forming one or more samples, (c) dispensing one or more samples into one or more vessels, (d) incubating for 7 days at 37 C in a humidified atmosphere with 5% CO 2 and 5% O 2 , (e) performing an ATP standard dose response and measuring luminescence, (f) measuring relative luminescence of the one or more samples, (g) conversion of relative luminescence units of the one or more samples to standardized ATP units and (h) correlating the standardized ATP units to engraftment and multilineage reconstitution potential.

The invention relates to a kit for determining engraftment and multilineage reconstitution potential of cells and/or tissues wherein the kit encompasses (a) preparing one or more cell suspensions from the cells and/or tissues, (b) adding the one or more cell suspensions to a pre-dispensed and pre-mixed tube comprising a serum mix, a methyl

cellulose mix, a growth factor mix, a medium, an ATP-releasing reagent, and an ATP luminescence-monitoring reagent thereby forming one or more samples, (c) dispensing one or more samples into one or more vessels, (d) incubating for 7 days at 37 C in a humidified atmosphere with 5% CO 2 and 5% O 2 , (e) performing an ATP standard dose response and measuring luminescence, (f) measuring relative luminescence of the one or more samples, (g) conversion of relative luminescence units of the one or more samples to standardized ATP units and (h) correlating the standardized ATP units to engraftment and multilineage reconstitution potential. The kit provides any of the herein disclosed and instructions for performing the herein-disclosed methods. The cell suspension may comprise colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), high proliferative potential stem and progenitor cell (HPP-SP), burst-forming unit erythroid (BFU-E), granulocyte-macrophage colony- forming cell (GM-CFC), megakaryocyte colony-forming cell (Mk-CFC), T cell colony- forming cell (T-CFC), B cell colony-forming cell (B-CFC), colony-forming cell-blast (CFC- blast), high proliferative potential colony forming cell (HPP-CFC), macrophage colony- forming cell (M-CFC), granulocyte colony forming cell (G-CFC), colony-forming unit- erythroid (CFU-E), colony-forming cell-basophil (CFC-Bas) or colony-forming cell- eosinophil (CFC-Eo) or any combination thereof.

One or more growth factors may be added to the one or more samples. For example, the cell suspension may comprise B-CFC and the growth factor may comprise DL-7. If a growth factor is added, then a background control that does not contain the one or more growth factors is contemplated.

The cell suspension may be derived from normal blood, peripheral blood, bone marrow or umbilical cord blood. In another embodiment, the one or more cell suspensions may be thawed from frozen cell suspensions.

In one embodiment, one or more cell suspensions may comprise one or more stem cell subsets. The one or more stem cell subsets may include CD34, CD 117, CD 133 and CD90.

In another embodiment, the one or more cell suspensions may have a concentration of about 0.5 x 10 6 cells/ml or about 0.5 x 10 3 cells/100 μl culture, hi another embodiment, the one or more cell suspensions may be purified to CD34 + cells and may have a concentration of about 1 x 10 4 cells/ml to about 2 x 10 4 cells/ml or about 1 x 10 2 cells/ 100 μl culture to about 2 x 10 2 cells/100 μl culture.

The invention further provides for an additional incubation step after the 7 day incubation wherein the one or more samples may be transferred to a 23 C humidified incubator with 5% CO 2 and left to equilibrate at least 15 to 30 minutes, advantageously 15 minutes. In one embodiment, the serum mix may comprise bovine serum albumin, an insulin, an iron-saturated transferrin, a serum and MDM. hi another embodiment, the growth factor mix may comprise at least one growth factor selected from the group consisting of erythropoietin, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L, and leukemia inhibitory factor, and combinations thereof.

In one embodiment, the methyl cellulose mix may be between about 1.5% and about 2.5% methyl cellulose. In anther embodiment, the concentration of fetal bovine serum in the cell growth medium may be between about 0% to about 10% by volume. In yet another embodiment, the concentration of methyl cellulose in the cell growth medium may be about 0.7% by weight.

The invention also provides contacting the target cell population with at least one cytokine. The cytokine may be erythropoietin, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L, leukemia inhibitory factor, and combinations thereof.

In an advantageous embodiment, the reagent capable of generating luminescence in the presence of ATP may comprise luciferin and luciferase.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of and "consists essentially of have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, in which: FIG. 1 depicts the hematopoietic hierarchy, wherein all of the proliferating cell populations (all of the cells except the cells in the mature cell compartment) can be detected using HALO, if necessary, simultaneously;

FIG. 2 depicts the correlation between GM-CFC and CD34+ cells infused. 173 paired assays of GM-CFC and CD34 + performed during 1996 and 2000 on transplanted cells have been correlated. No correlation was found between the nucleated cell counts and either GM- CFC or CD34 (data not shown);

FIG. 3 depicts the classical colony- forming assay procedure;

FIG. 4 depicts a HALO kit for 4 x 96-well plates;

FIG. 5 depicts a comparison of luminescence decay between 1 st and 2 nd generation HALO platform;

FIG. 6 depicts an ATP dose response for a human BFU-E study on day 7 of culture;

FIG. 7A (left) depicts a time course comparison between HALO and manual enumeration for multipotential stem cells and erythropoietic and myelopoietic progenitor cell populations; FIG. 7B (right) depicts a time course comparison between HALO and manual enumeration for granulocyte, macrophage, magakaryocyte and T- and B-progenitor cell populations;

FIG. 8 A (left) depicts a correlation of day 7 RLU readings with day 10 manual cluster counts (cell dose response from 2,500 to 20,000 cells/well); FIG. 8B (right) depicts a correlation of day 7 RLU readings with day 14 manual cluster counts (cell dose response from 2,500 to 20,000 cells/well);

FIG. 9A (left) depicts a phenotypic analysis of cells removed from HALO cultures on day 13;

FIG. 9B (right) depicts a phenotypic analysis of lymphopoietic populations derived from HALO cultures after 13 days incubation;

FIG. 10 depicts a comparison of doxorubicin dose response from stem and progenitor cells using HALO or manual enumeration;

FIG. 11 depicts a comparison of 5-flurouracil dose response for stem and progenitor cells by HALO and manual enumeration;

FIG. 12 depicts two variations of a HALO "in process control" kit;

FIG. 13 depicts a species comparison of paciltaxel and vinblastine on CFC-GEMM;

FIG. 14 depicts an assay, species and cell population comparison;

FIG. 15 depicts a conversion of relative luminescence units to standardized ATP units, demonstrating a standardization of HALO;

FIG. 16 depicts manual counting and colony identification in the colony-forming assay;

FIG. 17 depicts manual counting of proliferation units within a colony of cells;

FIG. 18 depicts a time course comparison between HALO and manual enumeration for multipotential stem cells and erythropoietic and myelopoietic progenitor cell populations;

FIG. 19 depicts a correlation of HALO with the number of "proliferation units";

FIG. 20 depicts a cell dose response for human bone marrow populations;

FIG. 21 depicts a HALO multiple population response profile using fresh human bone marrow mononuclear cells (N= 22, except for HPP-SP 1 where N= 7); FIG. 22A depicts the HALO SPC-QC single stem cell (CFC-GEMM) quality control protocol;

FIG. 22B depicts the HALO SPC-QC 2-stem cell quality control protocol;

FIG. 22C depicts the HALO SPC-QC 4-population quality control protocol;

FIG. 22D depicts the HALO SPC-QC 7-population quality control protocol; FIG. 23 depicts the effect of whole or lysed cord blood on HALO CFC-GEMM cultures;

FIG. 24 depicts the HALO-7 population response using umbilical cord blood (samples (N=8) processed using Hetastarch);

FIG. 25 A (top) depicts the growth potential of 7 populations present in 3 processed cord blood samples detected by HALO and

FIG. 25B (bottom) depicts the differentiation potential of 3 processed cord blood samples detected by differential counting of colonies derived from CFC-GEMM. DETAILED DESCRIPTION

The present invention provides kits that comprise vessels, each vessel containing one or more of the necessary reagents mixes and instructions for the use thereof for performing the high-throughput assays of the present invention. The kits and instructions of the present invention provide high-throughput assays for detecting and measuring the proliferative status of populations of cells, especially of primitive hematopoietic stem and progenitor cells, and cell lineages derived therefrom.

The hypothesis upon which this application is based in part, is that by non- subjectively detecting and quantitatively measuring the proliferation potential of several hematopoietic stem and progenitor cell populations simultaneously in a prospective or even retrospective manner, the 7 day assay can determine the "quality", engraftment, repopulation and multilineage reconstitution potential of the infused cells, thereby significantly increasing the ability to predict the transplant outcome and reducing risk to the patient.

The methods of the present invention are especially useful when applied to populations of primitive hematopoietic cells including primary cells isolated from peripheral blood cells, bone marrow cells, umbilical cord blood cells and hematopoietic stem and progenitor cells. The methods of the present invention, however, may be applied to any population of proliferating cells, including cells isolated from tissues and solid tumors.

The high-throughput assay methods of the present invention may also be used to determine the proliferative status of a population of hematopoietic stem or progenitor cells to determine the "quality", engraftment, repopulation and multilineage reconstitution potential of the infused cells and thus determine their suitability and acceptability for transplantation into a recipient animal or human patient.

The term "animal" as used herein refers to any vertebrate animal other than a human having a population of cells wherein at least one subpopulation of the cells may be proliferating or induced to proliferate. The term "animal" as used herein also refers to mammals including, but not limited to, bovine, ovine, porcine, equine, canine, feline species, non-human primates including apes and monkies, rodents such as rat and mouse, and lagomorphs such as rabbit and hare.

The term "tissue" as used herein refers to a group or collection of similar cells and their intercellular matrix that act together in the performance of a particular function. The primary tissues are epithelial, connective (including blood), skeletal, muscular, glandular and nervous.

The term "cell" or "cells" as used herein refers to any cell population of a solid or non-solid tissue including, but not limited to, a peripheral blood cell population, bone marrow cell population, a leukemic cell line population and a primary leukemic cell line population or a blood stem cell population. The cells may be hematopoietic cells, including bone marrow, umbilical cord blood, fetal liver cells, yolk sac and differentiating embryonic stem cells or differentiating primordial germ cells or embryonic germ cells. The cells may be a primary cell line population including, but not limited to, a leukemic cell line. Examples of leukemic cell lines include, but are not limited to, an acute lymphocytic leukemia, an acute myeloid

leukemia, a chronic lymphocytic leukemia, a chronic myeloid leukemia and a pre-B acute lymphocytic leukemia. Such cell lines include, but are not limited to, acute myelogenous leukemia, acute T-cell leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, acute monocytic leukemia and B-cell leukemia. The term "target cell population" as used herein refers to any cell population, especially hematopoietic stem and progenitor cells, or subpopulations thereof, that may be contacted with a test compound, wherein the test compound may modulate the proliferation of the cells in a positive or a negative direction depending upon the compound and the target cell population.

The term "cell line" refers to cells that are harvested from a human or animal adult or fetal tissue, including blood and cultured in vitro, including primary cell lines, finite cell lines, continuous cell lines, and transformed cell lines.

The term "cell lineage" as used herein refers to a cell line derived from a stem cell or progenitor cell that is committed to producing a specific functional cell including, but not limited to, mature cells of the a hematopoietic system. The term "cell cycle" as used herein refers to the cycle of stages in the replication of a eukaryotic cell. The cycle comprises the four stages Gl, S, G2 and M, wherein the S phase is that portion of the cycle wherein the nucleic acid of the cell is replicated. Thus, a cell identified as being in the S-phase of the cell cycle is also identified as being a proliferating cell. The term "proliferative status" as used herein refers to whether a population of hematopoietic stem or progenitor cells, or a subpopulation thereof, are dividing and thereby increasing in number, in the quiescent state, or whether the cells are not proliferating, dying or undergoing apoptosis.

The terms "modulating the proliferative status" or "modulating the proliferation" as used herein refers to the ability of a compound to alter the proliferation rate of a population of hematopoietic stem or progenitor cells A compound may be toxic, wherein the proliferation of the cells is slowed or halted, or the proliferation may be enhanced such as, for example, by the addition to the cells of a cytokine or growth factor.

The term "quiescent" refers to cells that are not actively proliferating by means of the mitotic cell cycle. Quiescent cells (which include cells in which quiescence has been induced as well as those cells which are naturally quiescent, such as certain fully differentiated cells) are generally regarded as not being in any of the four phases Gl, S, G2 and M of the cell cycle; they are usually described as being in a GO state, so as to indicate that they would not normally progress through the cycle. Cultured cells can be induced to enter the quiescent

state by various methods including chemical treatments, nutrient deprivation, growth inhibition or manipulation of gene expression, and induced to exit therefrom by contacting the cells with cytokines or growth factors.

The term "primary cell" refers to cells obtained directly from a human or animal adult or fetal tissue, including blood. The "primary cells" or "cell lines" may also be derived from a solid tumor or tissue, that may or may not include a hematopoietic cell population, and can be suspended in a support medium. The primary cells may comprise a primary cell line.

The term "primitive hematopoietic cell" as used herein refers to any stem, progenitor or precursor cell that may proliferate to form a population of hematopoietic cells. The term "hematopoietic stem cells" as used herein refers to pluripotent stem cells or lymphoid or myeloid (derived from bone marrow) stem cells that, upon exposure to an appropriate cytokine or plurality of cytokines, may either differentiate into a progenitor cell of a lymphoid or myeloid cell lineage or proliferate as a stem cell population without further differentiation having been initiated. "Hematopoietic stem cells" include, but are not limited to, colony- forming cell-blast (CFC-blast), high proliferative potential colony forming cell (HPP-CFC) and colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) cells.

The terms "progenitor" and "progenitor cell" as used herein refer to primitive hematopoietic cells that have differentiated to a developmental stage that, when the cells are further exposed to a cytokine or a group of cytokines, will differentiate further to a hematopoietic cell lineage. "Progenitors" and "progenitor cells" as used herein also include "precursor" cells that are derived from some types of progenitor cells and are the immediate precursor cells of some mature differentiated hematopoietic cells. The terms "progenitor", and "progenitor cell" as used herein include, but are not limited to, granulocyte-macrophage colony-forming cell (GM-CFC), megakaryocyte colony-forming cell (Mk-CFC), burst- forming unit erythroid (BFU-E), B cell colony-forming cell (B-CFC) and T cell colony- forming cell (T-CFC). "Precursor cells" include, but are not limited to, colony-forming unit- erythroid (CFU-E), granulocyte colony forming cell (G-CFC), colony-forming cell-basophil (CFC-Bas), colony-forming cell-eosinophil (CFC-Eo) and macrophage colony-forming cell (M-CFC) cells.

The term "cytokine" as used herein refers to any cytokine or growth factor that can induce the differentiation of a hematopoietic stem cell to a hematopoietic progenitor or precursor cell and/or induce the proliferation thereof. Suitable cytokines for use in the present invention include, but are not limited to, erythropoietin, granulocyte-macrophage colony

stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L, leukemia inhibitory factor, insulin-like growth factor, and insulin. The term "cytokine" as used herein further refers to any natural cytokine or growth factor as isolated from an animal or human tissue, and any fragment or derivative thereof that retains biological activity of the original parent cytokine. The cytokine or growth factor may further be a recombinant cytokine or a growth factor such as, for example, recombinant insulin. The term "cytokine" as used herein further includes species- specific cytokines that while belonging to a structurally and functionally related group of cytokines, will have biological activity restricted to one animal species or group of taxonomically related species, or have reduced biological effect in other species.

The terms "cell surface antigen" and "cell surface marker" as used herein may be any antigenic structure on the surface of a cell. The cell surface antigen may be, but is not limited to, a tumor associated antigen, a growth factor receptor, a viral-encoded surface-expressed antigen, an antigen encoded by an oncogene product, a surface epitope, a membrane protein which mediates a classical or a typical multi-drug resistance, an antigen which mediates a tumorigenic phenotype, an antigen which mediates a metastatic phenotype, an antigen which suppresses a tumorigenic phenotype, an antigen which suppresses a metastatic phenotype, an antigen which is recognized by a specific immunological effector cell such as a T-cell, and an antigen that is recognized by a non-specific immunological effector cell such as a macrophage cell or a natural killer cell. Examples of "cell surface antigens" within the scope of the present invention include, but are not limited to, CD3, CD4, CD8, CD34, CD90 (Thy- 1) antigen, CDl 17, CD38, CD56, CD61, CD41, glycophorin A and HLA-DR, CD133 defining a subset of CD34.sup.+ cells, CD 19, and HLA-DR. Cell surface molecules may also include carbohydrates, proteins, lipoproteins or any other molecules or combinations thereof, that may be detected by selectively binding to a ligand or labeled molecule and by methods such as, but not limited to, flow cytometry.

The term "cell surface indicator" as used herein refers to a compound or a plurality of compounds that will bind to a cell surface antigen directly or indirectly, and thereby selectively indicate the presence of the cell surface antigen. Suitable "cell surface indicators" include, but are not limited to, cell surface antigen-specific monoclonal or polyclonal antibodies, or derivatives or combinations thereof, and which may be directly or indirectly linked to a signaling moiety. The "cell surface indicator" may be a ligand that can bind to the

cell surface antigen, wherein the ligand may be a protein, peptide, carbohydrate, lipid or nucleic acid that is directly or indirectly linked to a signaling moiety.

The term "flow cytometer" as used herein refers to any device that will irradiate a particle suspended in a fluid medium with light at a first wavelength, and is capable of detecting a light at the same or a different wavelength, wherein the detected light indicates the presence of a cell or an indicator thereon. The "flow cytometer" may be coupled to a cell sorter that is capable of isolating the particle or cell from other particles or cells not emitting the second light.

The term "reagent capable of generating luminescence in the presence of ATP" as used herein refers to a single reagent or combination of components that, in the presence of ATP, will generate luminescence. The amount of luminescence may be reliably related to the amount of ATP present. An example of a reagent suitable for use in the present invention is the combination of luciferin and luciferase as described by Crouch et al. (J. Immunol. Meth. 160, 81-88 (2000)) and Bradbury et al. (J. Immunol. Meth. 240, 79-92 (2000) incorporated herein by reference in their entireties.

The term "toxicity" as used herein refers to the ability of a compound or a combination of compounds to negatively modulate the proliferation of a population of hematopoietic stem or progenitor cells. It will be understood that the toxicity of a compound or compounds may be effective against one hematopoietic cell lineage and not against another, and may further include the ability of a compound to modulate the differentiation of a hematopoietic stem or progenitor cell.

The term "differentially distinguishable" as used herein refers to hematopoietic stem and progenitor cells, or any other animal cell, the proliferation status of which may be usefully determined by the assay methods of the present invention and which can be characterized into subpopulations based on, for example, different complements of cell surface markers.

Abbreviations used in the present specification include the following: HALO, Hematopoietic and/or Hematotoxicity Assays via Luminescence Output; IL, interleukin; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline (10 mM phosphate, 138 mM NaCl, 2.7 mM KCl, pH 7.4); FBS, fetal bovine serum; BSA, bovine serum albumen; BITSI, (B)ovine serum albumin, recombinant human (I)nsulin, iron- saturated (T)ransferrin, (S)erum and (I)MDM; DVIDM, Iscove's modified Dulbecco's medium.

The high-throughput assay methods of the present invention comprise determining the proliferative status of a target cell population by measuring the metabolic activity of samples

of proliferating cells as indicated by their ATP content. The ATP content can be measured by detecting the luminescence generated by an ATP-dependent reaction requiring, for example, by contacting the cells with an ATP-releasing agent and an ATP luminescence-monitoring agent. A suitable system for detecting ATP by the emission of luminescence comprises the combination of luciferin and luciferase, although it is contemplated that any method that will emit a detectable signal, the intensity of which may be correlated to the amount of ATP in a cell culture may be within the scope of the present invention.

The HALO platform of the present invention provides the biotechnology and pharmaceutical industry with a rapid, high-throughput, multifunctional testing system that can be used at all stages of drug development from screening to clinical trials. HALO is a proliferation assay with a luminescence readout typically performed in a 96-well plate. The present invention provides kits that allows the detection of different stem, progenitor and precursor cell populations, from different hematopoietic tissues, to be detected and quantitatively measured simultaneously. Primitive hematopoietic cells can be isolated from suitable animal or human tissues including, for example, peripheral blood, bone marrow, or umbilical cord blood. Mononuclear cells, for example peripheral blood mononuclear cells (PBMCs) may be further isolated by methods such as density-gradient centrifugation. It is contemplated to be within the scope of the present invention for the primitive cell population to be further subdivided into isolated subpopulations of cells that are characterized by specific cell surface markers.

The high-throughput assay methods of the present invention are also suitable for screening a population of hematopoietic stem or progenitor cells to determine the proliferation status of the cells or subpopulations thereof wherein the proliferative status will indicate the suitability of the stem or progenitor cells for transplantation into a recipient animal or human host. The high-throughput assay of the present invention will allow the selection of populations of primitive hematopoietic cell that will likely proliferate and maintain engraftment within the recipient patient.

The population of primitive hematopoietic cells comprises at least one stem cell lineage selected from the group consisting of colony-forming cell-blast (CFC-blast), high proliferative potential colony forming cell (HPP-CFC) colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM).

In another embodiment, the population of primitive hematopoietic cells comprises at least one hematopoietic progenitor cell lineage selected from the group consisting of granulocyte-macrophage colony-forming cell (GM-CFC), megakaryocyte colony-forming

cell (Mk-CFC), macrophage colony- forming cell (M-CFC), granulocyte colony forming cell (G-CFC), burst- forming unit erythroid (BFU-E), colony-forming unit-erythroid (CFU-E), colony-forming cell-basophil (CFC-Bas), colony-forming cell-eosinophil (CFC-Eo), B cell colony-forming cell (B-CFC) and T cell colony-forming cell (T-CFC). If tissues are processed so that hematopoietic stem cells are transplanted, then it follows that the in vitro GM-CFC progenitor cell is not the cell population of choice for stem cell quality or engraftment, but rather the in vitro multipotential stem cell population or CFC- GEMM. The in vitro multipotential stem cell population, CFC-GEMM, is a prime candidate since its presence and growth potential can be detected and measured in 7 days using HALO. Committed progenitor cell populations are also detected after 7 days in culture. Therefore the CFC-GEMM would be one of 2 stem cell population incorporated into the kit.

Hematopoietic commitment into the erythropoietic, myelomonocytic and megakaryocytic pathways can be detected by the presence of the first committed descendants of the CFC-GEMM population, namely the GM-CFC, BFU-E, Mk-CFC progenitor cell populations. By definition, the CFC-GEMM population gives rise to cells in these hematopoietic lineages, and therefore provides an indication as to whether the quality of the stem cells allows production of lineage-specific cells. That balanced and not lopsided commitment occurs would be determined by the presence of the GM-CFC, BFU-E and Mk- CFC. Since the formation of lymphopoietic cells is dependent on the presence of primitive cells, it follows that the inclusion of a more primitive stem cell population than the CFC- GEMM, would be beneficial to the predictive value of the kit. However, the second stem cell population is more problematical for several reasons. On the one hand, the more primitive the stem cell, the greater the quiescent state, i.e. fewer cells in cycle. On the other hand, when stimulated, the more primitive the stem cell, the greater the proliferative potential and the larger the colony, but the longer it takes to be detected. However, detecting such a primitive stem cell population would predict that long-term repopulation cells are present in the transplant. Prior to finalizing the make-up of the kit, a decision has to be made regarding whether a second stem cell population can be included and if so, which population it will be and how long it will take to be detected using HALO technology.

The growth factor Flt3-ligand has been shown to expand and activate NK cells as well as dendritic cells and other T-cell populations. As mentioned above, one of the stem cell populations that can be measured with the HALO kit is designated CFC-GEMM 3, which is stimulated with EPO, GM-CSF, G-CSF, IL-3, IL-6, SCF, TPO and Flt3-ligand. Changing the

growth factor combination to include IL-3, IL-6, G-CSF, GM-CSF SCF and Flt3-ligand has been shown to induce the equivalent of the murine HPP-CFC cell population for human cells. Originally discovered in 1979 by Bradley and Hodgson, the high proliferative potential colony- forming cell (HPP-CFC) has been used as a marker for primitive stem cells, both in basic and clinical research. The human equivalent was first detected in 1989 by McNiece et al, who used high doses OfGM- 1 CSF and IL-3 to produce these colonies. The hallmark of this population is the production of macroscopic colonies, several millimeters in diameter that take between 14 and 21 days to form. This population is also relatively insensitive or resistant to 5-fluorouracil (5-FU) indicating that it is not normally in cell cycle. The CFC-blast population would be a relatively easy population to detect if it were not for the fact that to verify this cell, primary colonies have to be removed and replated a second time. The long-term culture-initiating cell or LTC-IC, also called the cobblestone area-forming cell (CAFC), is the most primitive in vitro stem cell population that can be detected, but requires a large number of procedures and entails a complicated process requiring 5-7 weeks to perform. From a practical point of view, neither of these stem cell populations would be applicable to a kit format or for clinical purposes.

CFC-GEMM-3 and HPP-CFC populations may also be included. Many of the growth factors required are similar for both populations and the differentiating factor might be the time required for measurement. Nevertheless, one of the requirements for this population is to measure both hematopoietic and lymphopoietic cells and to this end, combinations of growth factors would be used that would stimulate both systems.

One growth factor combination includes GM-CSF, G-CSF, SCF, IL-3, IL-6, TPO and Flt-3 ligand, while another uses the same combination, but with IL-2. If results do not produce the required endpoints, then other additional growth factors, such as IL-I, IL-4, IL-7 and IL- 15 may be added

In an advantageous embodiment, the following basic protocol may be used. Mononuclear cells from target tissues are prepared using Leucosep tubes (Greiner Bio-One). These 15ml or 50ml have a porous filter disc or flit, which allows easy, rapid and above all, standardized separation of MNCs using a density gradient medium such as FicoU-Hypaque Plus (Pharmacia) or Nycoprep (Greiner Bio-One). After washing and resuspension in medium, the nucleated cell count is determined and an aliquot used for phenotypic analysis as described below. The cell concentration will be adjusted for culture and added to premixed tubes containing the serum mix, methyl cellulose mix and growth factor mix to be tested, to produce a master mix. This is divided into wells of a 96-well plate containing 104 μl of the

master mix. A measure of proliferation by luminescence is performed only after full growth of the colonies is attained. Furthermore, since the cell composition of the colonies is also required, sufficient cell replicates are used to determine both proliferative status and phenotypic analysis. To obtain sufficient cells for phenotypic analysis, 8-12 replicates are prepared specifically for this purpose and another 8-12 replicates to measure proliferative status by luminescence. This means that 16-24 wells will be available for manual enumeration of the colonies.

The results from phenotypic analysis studies shown in FIGS. 9 A and 9B were performed by adding PBS to each well and mixing to reduce the viscosity of the methyl cellulose. This was repeated several times so that the cells could eventually be centrifuged and resuspended in PBS .and incubated with fluorochrome-'conjugated antibodies to different surface markers. To perform several phenotypic panels, all of the cells were pooled and then aliquoted into different tubes. Each of the tubes containing different antibody panels were analyzed using an EPIC XL/MCL flow cytometer. With large numbers of wells, this is a tedious and time-consuming process. This and other techniques will be significantly improved by the acquisition in the second half of 2004 of a Beckman Coulter FX500 flow cytometer capable of using a 96-well plate formation. Depending on the number of antibody panels required, the phenotypic composition of cells present in each well are analyzed. If 12 replicate wells are available and 6 antibody panels are to be measured, each panel can be performed in duplicate and the whole process, semi-automated. Using a liquid handler, PBS is added and the well contents mixed. After centrifugation of the plates, the antibody cocktails is added and after a 10 minute incubation period, ImmunoPrep reagents (Beckman Coulter) which lyse red blood cells, stabilize and fix the remaining cell suspension is added. The phenotypic composition of the cells in each well is then acquired and analyzed. All panels used contain the CD45 pan-leukocyte membrane marker. Phenotypic analysis include antibodies to detect the presence of those markers shown in FIGS. 9A and 9B, as well as CD90 (Thy 1 antigen present on primitive stem cells), HLA-DR (histocompatability antigen) and CD38 (progenitor cell lineage marker). Panels consist of 4 antibodies and the absolute number of each cell population will be obtained using beads which allow conversion to a concentration per μl of sample. The information obtained from phenotypic analysis must show that cells from all 8 lineages are present. This, together with manual and luminescence enumeration allows a decision to be made on the growth factor combination required for inclusion into the final kit. However, to determine the optimal time to measure luminescence

and to ensure that the stem cell population is sufficiently primitive to determine long-term repopulation potential, 2 other studies have to be performed.

For the chosen growth factor combination, a similar time course study to that shown in FIG. 7 will be necessary. The aim is to determine the optimal time to measure luminescence. For this, cultures in 96-well plates will be prepared so that 1 plate can be terminated on each day of the incubation period. Sufficient replicates will also be prepared so that both luminescence and phenotypic analysis can be performed. On each day, the number of PUs and colonies (if present) will be counted manually. Half of the replicates will then be subjected to luminescence measurement while the other half will undergo phenotypic analysis. There are 3 possible types of growth curve. The first would be similar that that obtained in FIG. 7A in which exponential proliferation followed by a decrease would be observed. The second might be a continuous proliferation as seen for many of the populations in FIG. 7B. The third might be a biphasic proliferation curve in which the first phase would be an initial proliferation phase, followed by a second, but greater proliferation phase that would produce the large numbers of cells required to make up a macroscopic colony. In the former case, a time point would have to be used that would ensure the presence of early non-committed cells, while in the second case, this would be later. If the third case were to occur, then the optimal time to measure luminescence would be during this second phase of proliferation. Long-term repopulation requires that primitive stem cells are present and, by definition, the stem cells exhibit extensive self renewal potential. This means that if stem cells with different degrees of "sternness" are present, the more mature cells would exhibit limited self-renewal capacity, while the more primitive would exhibit greater self renewal capacity. To ensure that cells are present with greater self-renewal capacity, replicate cultures are prepared and at a time at which proliferation is optimal and/or maximum, the cells from individual wells are removed and replated into secondary wells under the same conditions. The growth of secondary cultures and the presence of phenotypically positive stem cells, implies that primitive stem cells were present in the initial culture and that long- term repopulation potential can be predicted. This would define the growth factor combination to be used for the population being measured, and the optimal time for luminescence measurement.

HALO can be used to detect and measure multiple lineages simultaneously. In addition to these 9 cell populations of FIGS. 7 A and 7B, the HALO Kit has been designed so that several other populations can be measured. Besides basic CFC-GEMM, designated

CFC-GEMM-I, by adding TPO in addition to EPO, GM-CSF, G-CSF, IL-3, IL-6 and SCF, a CFC-GEMM-2 population has been added. By incorporating Flt3 -ligand to the 7 growth factor combination, a third subset designated CFC-GEMM-3 has been included. With the addition of F13-ligand, a slightly more primitive stem cell populations is stimulated than either CFC-GEMM 1 or 2. There are 2 more primitive stem cell populations that are being developed for HALO. The most primitive is the colony-forming cell ~ blast (CFC-blast) followed by the high proliferation potential colony- forming cells (HPP-CFC). The kit developed for stem cell transplantation quality control include 4 progenitor cell populations and 2 stem cell populations. At least 1 of the stem cell populations measured will be CFC- GEMM-2. This would provide information regarding short-term reconstitution. The other stem cell population would be either CFC-GEMM-3 or HPP-CFC. Using HALO, CFC- GEMM-3 population is measured at the same time as the other multipotential stern cell populations. However, HPP-CFC is a more primitive population and would therefore be particularly useful to predict long-term reconstitution. The majority of work performed using the HALO Platform has been to examine the toxicity of compounds on stem and progenitor cells of the blood-forming system using human as well as animal bone marrow target cells. HALO has been used for this purpose as described in Example 1 and shown in FIGS. 7, 10, 11, 13 and 14 and is contemplated in the methods of the present invention. The cells are mixed and suspended in a master mix containing (1) fetal bovine serum

(FBS) and optionally, serum supplements, (2) methyl cellulose and (3) growth factor mixes that stimulate different lympho-hematopoietic cell populations. Target hematopoietic stem and/or progenitor cells may be isolated from animal or human tissues and suspended at cell concentrations ranging from about 1 x 10 2 to about 2 x 10 5 /ml. Since typical assay volumes are 100 μl, actual cell concentrations in the assay test vessels may be diluted to 1/10 of the original starting cell concentration, hi one embodiment, the cells are mixed and suspended in methyl cellulose containing 0% to about 30% concentration of fetal bovine serum (FBS), 1% detoxified bovine serum albumin (BSA), iron-saturated human transferrin at a final concentration of 1 x 10 "10 mol/L, O-thioglycerol at a final concentration of 1 x 10 "4 mol/L and cytokines/growth factors. The methyl cellulose concentration in the assays of the present invention is between about 0.4% and about 0.7%, with a preferred concentration for most cell populations of about 0.7%. One exemplary medium is Iscove's Modified Dulbecco's Medium (IMDM, Life Technologies, Rockville, Md.) although other suitable media capable of supporting the growth of hematopoietic cells may also be used. Low fetal bovine serum

concentrations of between 0% and 10% can also be used. When the assay methods of the present invention are used under serum- free conditions, insulin (10 μg/ml) and, where necessary, low density lipoproteins (40 μg/ml) can replace the FBS.

For example, in one embodiment of the present invention, Iscove's Modified Dulbecco's Medium (IMDM) obtained from Invitrogen/Gibco (Carlsbad, Calif.) is prepared in small amounts (100-150 ml) using sterilized, 17.3 MOhm water. A 1.75% methyl cellulose stock solution containing alpha-thioglycerol was prepared in IMDM. The volumes of all reagents are dependent upon the final volume(s) required for the study. The final volume of reagents is, in turn, dependent on the amount of methyl cellulose that can be dispensed in multiples of standard volumes using a repeater syringe. The components may be dispensed into tubes using electronic pipettes as follows: 5% FBS, 20% BIT, growth factors, methyl cellulose and IMDM. The components are thoroughly mixed on a vortex mixer and cells are added and mixed again. The tubes are centrifuged briefly to 500 rpm so that the components are removed from the walls of the tubes. 100 μl of reagent mix is dispensed into replicate wells of a white, multi-well plate. The luminescence plate has a clear base so that cell growth can be observed under the inverted microscope.

Culture plates are incubated at 37 Celsius in a humidified atmosphere containing 5% CO 2 and 5% O 2 . On the day of analysis, the plates are transferred to a humidified incubator with 5% CO 2 at 22 Celsius to equilibrate. Using a multichannel pipette, 125 μl of ATP-releasing reagent (ATP releasing reagent) is added to each well, mixed and returned to the 22 degree Celsius incubator for 15 mins. Thereafter, 20 μl of ATP luminescence-monitoring reagent (ATP luminescence- monitoring reagent) is added and the luminescence read immediately. Data from the plate reader is used to calculate the mean, standard deviation and percent variation automatically for graphical presentation and/or statistical evaluation respectively. For all assays, a 10 μM ATP standard can be performed on the day of analysis to provide quality control for the reagents and equipment as well as a reference to which all values can be calculated. The high-throughput assay method of the present invention further includes contacting a hematopoietic stem or progenitor cell population with at least one cytokine that can induce the proliferation of the stem or progenitor cell population. The cytokine, or a combination of cytokines, maybe selected to induce the differentiation and proliferation of selected subpopulations of, for example, hematopoietic cell lineages. Exemplary cytokines include, but are not limited to erythropoietin, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor,

thrombopoietin, stem cell factor, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L, leukemia inhibitory factor. Additional growth factors, alone or in combination, may also be included to boost the proliferative status of a particular culture of cells, including such factors as insulin-like growth factor, insulin and recombinant insulin.

The stock cell culture is aliquoted into sample chambers. While sample chambers may be the wells of a multi-well tissue culture plate, and preferably a or 96-well plate, it is also contemplated to be within the scope of the present invention to conduct the assays of the present invention in any other suitable reaction vessels including, but not limited to, individual tubes, wells of plates and the like. Culture plates with a well surface area of about 35 mm 2 and a low ring of about 2 mm high are especially useful and allow colonies to be counted that are against the wall of the ring. Preferably the sample chambers are not tissue culture treated.

Plastic that is sterilized and tissue culture treated exhibits different surface properties than plastic that is not sterilized by radiation and not tissue cultured treated. The change in surface properties results in cells preferentially adhering to the plastic and growing more rapidly than colony-forming cells. This is especially true if the cell suspension contains macrophages and other microenvironmental cell components. Also, the surfaces of individual wells of a multi-well plate may not be treated homogenously and may result in complete growth inhibition in a significant number of wells. This can be a random event such that although 8-12 replicates may have been plated, up to 5 wells on a single plate might exhibit no growth whatsoever. Unwanted preferential adherence and growth may be avoided by using "non-sterile" and untreated plates. All tissue culture articles made from "virgin" plastic under very high temperatures, when released from the mold, are sterile. Contamination problems are unlikely. Non-treated and non-irradiated plates allow superior growth for the methods of the present application.

For luminescence assays to be performed, multi-well plates that reduce background light emission or scatter when the plates are being enumerated in the plate reader may also be used. While it is desirable to use replicate reactions, it is to be understood that a single reaction sample may be used for determining the proliferative status of cells for each data point. However, replicate reactions are to be preferred wherever an increase in accuracy is necessary. For example, reactions may be replicated once, twice or more times, including on a single multi-well plate, although quadruple reactions are preferred.

The cultures can be incubated in a humidified atmosphere having a low oxygen tension for a period preferably extending to about 10 days but also to at least about 14 days. A suitable oxygen concentration range is from about 3.5% oxygen to about 7.5% oxygen, most preferably about 5.0% oxygen, and further comprising about 5% CO 2 as described by Bradley et al. (J. Cell Physiol. 97, 517-522 (1968) and Rich & Kubanek (Exp. Hemat. 52, 579-588 (1982) incorporated herein by reference in their entireties.

In the assay methods of the present invention, the culture conditions include D- thioglycerol to maintain molecules in a reduced form, and the cultures are incubated under low oxygen tension of between about 3.5% oxygen and about 7.5% oxygen, both conditions reducing oxygen toxicity. The cell aggregate or colony can be maintained in a stagnant or non-proliferative state for between about 2 and about 3 weeks. Other cells, however, that are developmentally more primitive, for example, stem and progenitor cells, have a greater proliferative capacity and will begin to form colonies after a certain lag period of time. These cells will continue to divide throughout the whole of the incubation period. Eventually, the proliferative capacity of the cells within these colonies will also decrease and finally cease.

The HALO platform is based on a non-subjective luminescence output rather than subjective manual enumeration. Two types of luminometers have been used. The original instrument was a relatively inexpensive TECAN GENios plate reader with Magellan software, capable of measuring absorbance, fluorescence and luminescence. A more recent acquisition has been a dedicated Molecular Devices Lmax luminometer with Softmax Pro software. The luminescence assay is a two step process by first releasing intracellular ATP and then measuring it according to the following equation:

Luciferase ATP + Luciferin + O 2 π Oxyluciferin + AMP + PPJ; + CO 2 + LIGHT Mg 2+

The first step is to release ATP from the growing cells using an ATP releasing reagent (ATP-RR). After mixing the cultures with 20μl of this reagent followed by an incubation time of 15 min at 23 0 C, 1000 of an ATP monitoring reagent (ATP-MR) is added. Addition and mixing of the reagents is by electronic multichannel pipettes and for most kit users, this will be the preferred and most economical method. Manual addition and mixing of reagents can been replaced by a Beckman Coulter BioMek 2000 liquid handler. This has not only allowed high-throughput processing of the 96-well plates, but a drastic reduction in well-to- well variability. The original assay system used an ATP-MR that required an immediate luminescence reading. This has now been replaced by a reagent that allows a reading to be

completed during the first 30 min without significant decay. The effect of the 2 different reagents is shown in FIG. 5. Whereas the GENios instrument measures "glow type" luminescence and requires manually setting various parameters, including the gain, the Lmax can measure both "flash" and "glow" type luminescence and does not require any parameter settings. However, no two machines are alike and no standardization exists in luminescence measurements. For this reason, the term relative luminescence unit or RLU is used. For both internal studies and the kit, an ATP standard dose response is performed prior to sample measurement. This has 3 functions. First, it ensures that the luminometer is functioning correctly. Second, it ensures that the luminescence reagents are working well and have not deteriorated. Finally, it allows conversion of non-standardized RLU to a standardized ATP unit (in μM), so that results within and between laboratories can be compared over time. In short, HALO is a standardized colony- forming assay. FIG 6 shows a typical ATP standard dose response performed prior to every study. The ATP standard, ATP-RR and ATP-MR are on the right side of the kit (FIG. 4). Once the more primitive stem cell population (designated stem cell-1) has been defined in the above studies, the new kit can be designed. There are 2 possible configurations for a kit of this kind to be used in a potential clinical situation. The first would be similar to the HALO research kit shown in FIG. 4. The left side of the kit contains the 3 mixes required to culture the cells. The latter are provided by the user. The right side of the kit contains the ATP standard and the reagents for measuring luminescence. The growth factor combinations (shown in FIG. 4 as separate vials), in this case, for each of 4 populations to be measured, are added in the required ratios with the cells, mixed together and the master mix dispensed into the wells using a repeater syringe. Although this is an easy procedure, there is an even easier and more rapid way of performing this task, which has been alluded to above. All of the reagents for each of the cell populations to be measured would be provided in pre-made, pre- mixed individual tubes. The user only has to add the cells, mix and dispense the master mix into the wells. This is more efficient, saves time and is even more robust and reliable since the amount of pipetting needed is reduced to a bare minimum. There would, however, be 2 variations on this theme determined by the number of replicates used and populations tested. FIG. 12 shows these 2 variations. In variation 1, 8 replicates for each population are prepared and plated in columns from A to H. If 6 populations (Stem cell 1, CFC-GEMM-2, BFU-E, GM-CFC, MK-CFC and T-CFC) are detected, 2 samples are accommodated on each plate. In the second variation, 6 replicates are plated in rows from 1-6 and 7-12. However, in this case, 8 populations from 2 samples can be accommodated on 1, 96-well plate. Besides the 6

populations given above, the B-cell progenitor, B-CFC stimulated with EL-7, ,may be added, and a background could also be included to which no growth factors are added. The background is an important control, since it measures the ability of cells to spontaneously proliferate in the absence of growth factors and provides a value above which all other populations should exhibit luminescence. By including the background, it is then possible to arbitrarily define the state of proliferation. For example, if the RLU or ATP values for a population are greater than 2-3 times the standard deviation of the background, proliferation can be said to have occurred. An extra, non-sterile plate would be provided in the kit to perform the ATP standard dose response. Values for each population from each tissue would be provided to the user in the manual as a historical range that would be obtained, in part, from the results as derived.

Base line data each of normal and mobilized peripheral blood, bone marrow and umbilical cord blood using the new version of the HALO Kit will be obtained and the results will be compared with those obtained at the same time using the classical colony- forming assay.

An embodiment of the present invention is to validate the newly developed HALO Kit against the conventional colony-forming assay procedure. Another embodiment is to accumulate base-line data for normal tissue. Yet another embodiment is that once validation of the HALO Kit has been performed, it can then be used for further studies. A minimum of 20 samples of normal and mobilized peripheral blood, bone marrow and umbilical cord blood should be assessed. The tissues may be obtained from a commercial vendor, e.g. Cambrex/Poietics BioSciences, Walkersville, MD. The power statistic of this number of samples is 0.95 if the alpha value is 0.05 (PO.05). If more samples are available, these will also be analyzed. The data obtained will be included as normal historical values in the manual that will accompany the new kit.

There are, in fact, at least 5 alternative embodiments. The different alternatives are dependent on how the classical colony-forming assay is carried out. The HALO version defined above would remain the same throughout.

Alternative 1 : HALO is performed as described above using whatever version is deemed suitable. The CFA would be carried out in a traditional manner (FIG. 3A), by adding all the reagents, including the growth factors separately so that the total volume would be sufficient for 2-3 replicates of 1 ml each (total volume prepared 3.5-4 ml). Standard 1 ml Petri dishes would be used. After incubation, the cluster/colony count would be evaluated manually for the whole plate. The disadvantages to this technique is that large quantities of

culture reagents are used, which also increases the cost of each assay, and that setting up the assays would take considerable time, especially if large numbers of samples are to be analyzed at the same time.

Alternative 2: The second alternative is to use ready-made reagents provided by Stem Cell Technologies (Vancouver, BC), instead of adding the reagents separately. The problem with this is that reagents for each of the cell populations to be tested, namely stem cell-1, CFC-GEMM-2, GM-CFC, BFU-E, Mk-CFC, T-CFC and possibly B-CFC are not all available in the same format, so that most cell populations would have to be prepared as in the first alternative. Alternative 3 : In this case each CFA assay would be prepared for quadruplicate cultures of IOOI 11 each (total culture volume of 600 ml) as shown in FIG. 3B. The plates used in this micro-CFA procedure and previously developed are 1 ml Petri dishes which have 4 wells each with a surface area of 95.03 mm 2 . The clusters/colonies are counted in each well. Although the quantities are significantly reduced in this microculture method and would be the same volume/culture as that for HALO, the time to set-up the assay would be a limiting factor if many samples are obtained on the same day.

Alternative 4: The fourth alternative is similar to the third, but in this case, a single master mix is prepared so that sufficient volume is available for both the HALO and the 4- replicate, micro-CFA. Alternative 5: The last alternative is that a single master mix is prepared for each sample to be assayed on each of the cell populations, but both HALO and CFA and plated in duplicate 96-well plates using the same number of replicates. One 96-well plate would be counted for clusters prior to luminescence measurement on the designated day for HALO, while the other plate would remain in the incubator to produce mature colonies which would be counted before measuring luminescence. The difference in master mix volume between alternatives 4 and 5 would be between 400-500 ml. The difference in total volume would be 2.25ml for 16 wells and 2.0 ml for 12 wells. Despite the fact that both clusters and colonies are counted on different days, it usually takes between 1-1.5h to manually enumerate a 96- well plate, whereas it takes 3-5 times longer for Petri dishes. More important, however, is that the samples will all be handled in the same manner, for both types of assay, thereby reducing variability between the assays and increasing the significance between the assays. This is considered the best alternative because more comparable information can be obtained. Indeed, 4 parameters can be measured. These are manually enumerated cluster or PU number versus proliferation by luminescence and manual enumeration of differentiated cluster/colony

formation versus proliferation by luminescence at the mature colony time point. The ratio of mature colony numbers to luminescence values provides a direct indication of the amount of proliferation occurring during the differentiation phase. An analysis of variance (ANOVA) will allow correlation and statistical differences between the different parameter measurements to be ascertained. The prediction would be that similar correlation curves to those shown in FIGS. 8 A and B would be obtained for each population and tissue tested. Thus, the necessary validation would be obtained to allow HALO to be used in place of the classical CFA and provide the baseline values for normal tissues required for future clinical trial studies. The invention further provides for use of the HALO Kit to test clinical samples including (1) a HALO Kit to retrospectively test "blind", patient clinical samples to predict engraftment and multilineage potential and to compare the results with the patient clinical outcome at the end of the study and (2) use of the HALO Kit to compare samples from the same patients at one or more stem cell transplantation centers. Although the validation to use the new HALO Kit in place of the classical colony- forming assays is part of the invention, further validation uses patient samples. These are frozen samples from transplant donors. The samples are obtained from transplant centers and tested "blind". The results are compared with those of the transplanted patient to determine if the cells grew, engrafted and exhibited multilineage reconstitution. Use of the HALO Kit to compare samples from the same patients at one or more stem cell transplantation centers. Kits are prepared and distributed to one or more transplantation centers. Samples from the same patient would then be analyzed at all testing centers and the results compared. The goal is to demonstrate that the HALO Kit functions the same at all testing centers and that the results obtained are within acceptable ranges for each of the populations under study. This aim will pre-empt a larger clinical study that would be required as a basis for potential "clinical claims" that will be submitted to the FDA for review.

The newly developed HALO Kit are used to retrospectively test "blind", patient clinical samples to predict engraftment and multilineage potential and to compare the results with the patient clinical outcome at the end of the study.

This clinical study will not involve fresh "in process" quality control of tissue that is destined to be transplanted. The tissue to be used for this embodiment is frozen, processed samples that have, at some previous time point, been infused into a patient and for whom the clinical outcome is known. The clinical outcome will not be divulged during the course of

the study, but at the end, when all the in vitro HALO data have been accumulated and analyzed. Thus, HALO will be performed "blind". The only information is the origin of the original tissue, that is, normal or mobilized peripheral blood, bone marrow or cord blood and whether the tissue had been processed to the MNC stage or the CD34 + stage, since this will depend on the number of cells plated in vitro.

A minimum of 20 frozen patient/donor samples each of mobilized peripheral blood, bone marrow and umbilical cord blood is tested. This number provides the power statistic which would allow 95% confidence limits to be obtained. However, if time and funds permit, as many samples as possible will be processed in order to achieve better prediction statistics.

Preparation and the HALO procedure is described below.

Each frozen sample will be carefully thawed to avoid undue cell death and the release of DNA which can form clumps in the cell suspension and entrap viable cells, thereby reducing the nucleated cell count. If the cells have been frozen using dimethylsulphoxide (DMSO) as cryopreservant, one of two thawing methods is available: The first is to rapidly thaw the cells at 37°C and transfer the cells to PBS containing heparin and DNase. The second method also involves rapidly thawing the cells, but then all cells from the vial are transferred to an empty tube and a mixture containing PBS and serum at different volumes is added over a 10 min period. This timed DMSO dilution protocol is not usually the method used when frozen cells are transplanted. Normally the cells are rapidly thawed and infused almost immediately, although some toxicity from DMSO has been seen. It is possible that the presence of serum also results in clumping of the cells. An alternative protocol would be to perform a timed DMSO dilution procedure, but instead of using serum, heparin and DNase are used instead. Regardless of which procedure is used, the presence of DMSO is toxic in culture and its concentration must be reduced so that any cell growth is not masked. Cord blood cells are usually frozen in Hetastarch (HES) and thawed quickly at 37 0 C. As discussed in the above Examples, a SOP for sample thawing will be ascertained during these studies and might be different for each of the different tissues tested. This SOP will also help in the above Examples, where different transplantation centers will be performing the same procedures.

Once the cells have been thawed, washed and resuspended in 300-5000 of medium (Iscove's Modified Dulbecco's Medium, MDM), a nucleated cell count will be performed using a Beckman Coulter Z2 particle counter. A sample of the cell suspension will then be analyzed by flow cytometry for viability using 7- AAD as well as the presence of stem cell

subsets and the primary lineage-specific populations. The stem cell subsets will include CD34, CD 117, CD 133 and CD90. Content of CD34 + cells will be performed using the Beckman Coulter Stem Cell Kit and analyzed using the ISHAGE protocol. This relies on all CD34 + cells being CD45 + . If the sample has been designated to contain CD34 + cells, the CD34 analysis will determine whether the sample contains only CD34 + cells or whether other CD45 + /CD34 + cells are present. If the latter is the case, then other antigens will be determined. These will include glycophorin-A (erythroid cells), CD 14 (monocytes), CD 15 (neutrophils), CD41 ICD61 (megakaryocytes), the T-cell markers, CD3, CD4 and CD8 and the NK and B-cell markers CD56 and CD 19 respectively. All antibodies will be used in panels of 4, one of which will be the pan-leukocyte antibody to CD45, through which all other antibodies will be gated. Between 1 /xl and 20μl of sample will be used for each panel, but if insufficient cells are available for culture, then the number of panels will be reduced and priority given to using cells for HALO. The reason for performing phenotypic analysis is .to ascertain the composition of the starting cell suspension, so that it can be related to populations determined by HALO .

The majority of the cells suspension will be adjusted to the cell concentration required for cell culture. Normally, if only MNCs are available, then the cell concentration required would be about 0.5 x 10 6 cells/ml or about 0.5 x 10 4 cells/lOOμl culture. If the starting cell suspension has been purified to CD34 + cells, the starting concentration is reduced 10-100 fold. The appropriate cell concentration in a designated volume will be added to each tube containing the pre-mixed cultured reagents for the cell populations to be measured according to the configuration determined above (see, e.g., FIG. 12). After incubation at 37°C in a humidified atmosphere containing 5% CO 2 and 5% O 2 for the designated period of time, the plates are transferred to a 23 0 C humidified incubator with 5% CO 2 and left to equilibrate at that temperature for at least 30 min. Thereafter, the plates will be placed in the liquid handler and the ATP releasing reagent and monitoring reagent added and mixed with the culture components automatically. Finally the plates will be read in the Lmax luminometer and the data stored. Normally the results are exported to a text file which is then imported to an Excel spreadsheet, from where the results can be further analyzed using other software programs where necessary

A minimum of 20 samples from each tissue type is be analyzed. Thus a total of at least 60 samples is processed. Since HALO analysis always involves an ATP standard dose response, all RLU values will be converted to ATP (μM) values. Variation in the daily ATP standard dose response does occur due to use of a new batch of ATP and/or ATP-MR. In

order that samples tested at different times can be compared, a normalized ATP standard curve will be produced at the end of the study and all samples normalized against this standard curve, hi this way, day-to-day internal variations can be reduced and the response of the samples statistically analyzed and compared. From these data, the answers to the questions 1, 2, 3, 5, 7, and 9 for each sample will be tabulated. These predictive answers will then be compared with the clinical outcome for each patient sample by answering the questions 4, 6, 8 and 10. The hypothesis is that the in vitro data will predict the clinical outcome. If stem cells were present (Question 1) and they grew in HALO (Question 2), then engraftment should have taken place (Question 4). If Question 5 indicated that the stem cells became committed by the stimulation of CFC-GEMM, then the patient should have exhibited multilineage reconstitution (Question 6). If all lineages tested in HALO were present (Question 7), then it would be expected that a balanced or near normal multilineage reconstitution would have taken place (Question 8). If only certain progenitor cells grew in culture, then these lineages would be expected in the patient. If T-cells, were produced in culture (Question 9), then it would be expected that not only primitive stem cells would be present, but that either GvHD or GvL might have occurred (Question 10). IfHALO indicated that both short- and long-term stem cells were present (Question 3), then short- and long-term engraftment should have occurred and the patient might still be alive (Question 11) barring other eventualities. The samples used for this aim would be frozen, although if freshly processed tissue was made available, it would be incorporated into the study. The number of samples available for testing at several sites might also be a limiting factor, since sufficient samples have to be kept for regulatory purposes. It is important that as little information about the sample and patient be known at the time of testing. Therefore, to avoid any bias, the center providing the sample would distribute the sample to other testing centers, but would not test the sample themselves. This assumes that more than 1 transplant center would be involved in the study.

To test the samples at all facilities, they will have to be shipped by overnight courier. The participating centers will have sufficient kit materials to perform the studies, especially if fresh samples are used.

It is imperative that the samples are treated in exactly the same manner at all facilities prior to addition to the tissue culture mixes. This means that if the samples are frozen, each facility testing the sample will thaw it and handle it according to a designated SOP to be determined prior to the start of this study by all concerned. Thawing and handling frozen

material is an important first step and that developed procedures will help define a consensus SOP. Not all centers have similar instrumentation to evaluate the nucleated cell count. However, cell counting is a procedure that is performed under standardized conditions in most transplantation centers and where possible, these conditions will be used at all facilities. Standardized phenotypic analysis of CD34 + cells and their viability would be according to the ISHAGE protocol. Similarly, SOPs will ensure that the adjusted cell concentration for in vitro HALO testing is performed in the same manner and that the same cell concentration is used for a specific sample at all testing facilities.

Despite an endeavor to try and ensure that all procedures are standardized, there are some aspects which will not come under this umbrella. One of the most important is how the cells are incubated. This may appear to be a minor point, but there is in fact a significant difference regarding whether cells are incubated under atmospheric (21%) or low (5%) oxygen tension conditions. Bradley et al first demonstrate the advantageous effect of culturing GM-CFC under low oxygen. In 1982, Rich and Kubanek demonstrated that erythropoietic cells exhibited a greater plating efficiency in culture under low oxygen tension and that the inclusion of reducing agents such as beta-mercaptoethanol, alpha-thioglycerol or even vitamin E and reduced glutathione, had an additive effect when cells were grown under low oxygen. All HALO cultures are incubated under 5% oxygen tension which is approximately equivalent to about 45mmHg. This is also the venous partial oxygen tension in many organs, including the bone marrow. The increased plating efficiency, resulting from reduced oxygen toxicity caused by free radicals, allows greater sensitivity and therefore increased number of colonies in the CFA and higher luminescence values. Tissue culture incubators are now readily available to allow cells to be grown under low oxygen tension. However, it must be expected that few stem cell transplant centers will have access to these tissue culture incubators. Tissue culture of the 96-well plates for HALO will probably be performed in normal incubators gassed with 5% CO 2 .

Rather than just indicating cell growth, HALO can provide more important information. The indication that cells will growth is also a prediction of engraftment. The configuration of the new HALO Kit will incorporate a detection system that will predict both short- and long-term engraftment, by allowing stem cells with different degrees of growth potential to be detected simultaneously. By including other more mature blood-forming cell populations, the kit can also be used to determine if some or all of the blood cell lineages are going to be produced in the patient.

The research HALO Kit Platform provides a more rapid, reliable, robust, non- subjective and standardized testing system with high-throughput capability than the classical colony- forming assay. In contrast to the latter, HALO also allows flexibility and versatility due to its ability to detect and measure multiple cell populations from multiple species simultaneously. Accordingly, it is believed that the HALO Kit Platform is a superior product.

The invention also provides for a HALO Stem and Progenitor Cell Quality Control (SPC-QC) Platform which may be a replacement for the colony-forming assay for stem cell transplantation processing and cord blood storage. FIGS . 22 A-22D show the different configuration of the HALO SPC-QC platform.

For general screening of samples for growth potential, the single stem cell population (CFC- GEMM), with or without a control would be sufficient. For more advanced analysis of the sample, the 2-stem cell or 4-population kit could be used. To analyze all blood-producing lineages, the HALO 7-population kit would be used. This would provide possible predictive information regarding short- and long-term engraftment and repopulation potential prior to transplant.

From initial studies with cord blood, two factors interfere with HALO. The first is high concentrations of whole erythrocytes (FIG. 23), which provide oxygen causing an imbalance in the luciferin/luciferase reaction. The second is hemoglobin, released from lysed erythrocytes. This has been known to inhibit luminescence. FIG. 24 depicts the HALO-7 population response using umbilical cord blood. The results in FIG. 25 were obtained from three cord blood samples obtained from the Puget Sound Blood Center. The results indicate that the total number of colonies manually counted show a similar pattern of results to those obtained by HALO. Besides time and cost savings, HALO allows: (a) less skilled personnel required- does not rely on a single person, (b) time to proficiency: 1-2 weeks, (c) rapid, standardized, non-subjective and reproducible, (d) flexibility and expandability, (e) automated with high- throughput capability, (f) direct indicator of stem cell growth potential, (g) incorporation into proficiency testing and (h) may be predictive for long- and short term engraftment and reconstitution potential.

The invention will now be further described by way of the following non-limiting examples. EXAMPLE 1 : Hematopoietic/Hemotoxicity Assays via Luminescence Output (HALO)

Much of what is known today about the biology and physiology of the hematopoietic system has come from the use of the colony- forming assays. For example, the stem cell hierarchy and the effect of most of the growth factors and interleukins would not have been possible without this technique. The use of antiproliferative agents and other drugs, both in animal in vivo models and in vitro using the CFA, have helped elucidate how the hematopoietic system is regulated. Yet the CFA had never been used routinely during drug development to analyze and predict the effects of new drugs on stem and progenitor cells of the blood-forming system. The reasons are the same as those stated in the previous section, hi addition, the procedure was not capable of handling large numbers of compounds and testing them in multiple species and on multiple populations simultaneously. Conventional hemotoxicity testing for drug development only requires measurement of circulating blood parameters and morphology and/or pathology. As such, conventional hemotoxicity testing has little, if any predictive value.

AU of these aspects led to the HALO Platform, which was originally developed to provide the biotechnology and pharmaceutical community with a predictive, rapid, multifunctional in vitro system to test the effects of new drug candidates on stem and progenitor cells of the lympho-hematopoietic system.

The basis of both the CFA and HALO is the functional ability of cells that cannot be morphologically detected and are present in very low numbers, to be stimulated to grow in a semi-solid medium to produce clusters of proliferating cells which can differentiate into colonies (FIG. 3). Each cluster of proliferating cells and hence each colony is derived from a single stem, progenitor or precursor cell. The number of divisions the colony- forming cell can undergo is directly related to its proliferation capacity and hence its primitiveness or maturity and therefore the size of the colony produced. Originally, the semi-solid medium used was agar. Today, water-soluble methyl cellulose is used. As the cells proliferate and divide, they remain in place, due to the semi-solid immobilizing medium, and eventually form colonies over a period of time. It is these colonies that are manually enumerated under the microscope. In contrast to the classical CFA which is a functional differentiation assay, HALO is a proliferation assay; it measures the capacity of cells to enter the proliferation phase prior to differentiation. To enter the proliferation phase of growth, stimulators in the form of growth factors and interleukins are required. As the cells proliferate, there is a proportional increase in the intracellular ATP concentration. The HALO read-out depends on releasing the intracellular ATP, which them reacts with a luciferin/luciferase - containing reagent to produce bioluminescence in the form of light. This is, in turn, detected using a

luminometer. Since proliferation rather than differentiation is measured, the time required to obtain results is, for human cells, halved from 14 days to 7 days. In addition, HALO is performed in 96-well plates and the luminescence output can standardized using an ATP standard (see below). Thus, HALO has high-throughput capability and provides a non- subjective, standardized readout. Of particular importance for the present discussion is the ability to assay several different lympho-hematopoietic cell populations from different tissue sources side-by-side. For example, at least 14 different colony-forming cell populations from human peripheral blood, bone marrow and umbilical cord blood could be examined simultaneously. In short, HALO is an extremely powerful testing platform that has many applications, one of which is the ability to be used to determine the quality, engraftment and repopulation and multilineage reconstitution potential of stem cells transplanted into patients. Stem cell transplantation is now standard therapy for selected patients with leukemia, lymphoma and myeloma. According to the International Bone Marrow Transplant Registry (IBMTR), approximately 45,000 transplants were performed worldwide in 2001. These include autologous, allogeneic and cord blood transplants.

Most stem cell transplant centers now rely only on the viability and number of CD34 + cells and/or stem cell subsets. Hundreds of transplants have been performed using this type of data and one must assume that the majority have been successful, since the number of unsuccessful transplants is difficult to obtain. Yet unsuccessful transplants do occur. Table 1 shows the results. Two patients in a transplant center failed to engraft after receiving CD34 + separated mobilized peripheral blood cells. From the small aliquot of frozen cells made available, the absolute number of CD34 + cells and their viability was ascertained by flow cytometry and a growth potential assay using the conventional CFA technique was performed for the CFC-GEMM, GM-CFC, BFU-E and CFU-E populations (FIG. 1). The original infused volume was not provided so that the absolute number of CD34 + cells in this case has little meaning. Nevertheless, it is clear that even with relatively high numbers CD34 + cells and a viability above 60%, neither the multipotential stem cells (CFC-GEMM) nor the granulocyte-macrophage progenitor cells (GM- 1 CFC) grew. The presence of erythroid progenitors (BFU-E) and precursor cell (CFU-E) colonies, indicates not only skewed growth, but also that cell processing preferentially separated CD34 + progenitor cells. In any event, the outcome was that the patient did not engraft, because stem cells were not present, a situation that might have been predicted.

Table 1 : Lack of Engraftment and Skewed Repopulation after Mobilized Peripheral

Blood Transplant in 2 Patients

If it had been possible to examine the growth potential of cells prior to transplant, which can be done for mobilized peripheral blood or umbilical cord blood, then perhaps some of the unsuccessful transplants might have been averted and safety of the patient not compromised. Bone marrow transplants are more problematical since, as mentioned above, the bone marrow is infused soon after it is processed. Since the CFA takes 14 days until results are obtained and engraftment usually occurs between 14 and 21 days, the results are less likely to be of use. However, what if an assay, such as HALO, was available that could provide the results of growth potential within 7 days? If in vitro growth did not occur, this would provide the physician with more time (1-2 weeks) to organize either a second transplant or different therapy, if available. For both the physician and especially the patient, this provides an added safety net.

HALO can produce results that could predict engraftment and reconstitution of transplanted cells. The FDA has drawn a line between using the kit as an "in process control" and as a "clinical claim". An "in process control", has been defined as a quality control to ensure that the tissue being processed and the final product contain the cells that are supposed to be present. Besides this application of stem cell transplantation, processing, cryopreservation and thawing of umbilical cord blood for transplantation purposes is another direct application. A future application might occur when embryonic stem cells are used routinely for clinical applications. For an "in process control", no FDA submission and review is required. It follows, however, that if the final product is tested using the HALO platform for growth potential of stem cells, then the answer obtained will not only demonstrate that stem cells are present, but whether they will grow, since without growth, no stem cells can be present. If the stem cells grow in culture, then it is possible, by default, to predict that the stem cells will also grow and, therefore, engraft and repopulate the patient. According to the FDA, this would be a "clinical claim" and the kit would be subjected to the FDA approval procedure. If, in addition, to detecting stem cells in the final processed product, the presence of different progenitor cell populations were also detected, then it

would be possible to show that the stem cells are healthy and have the potential to be committed into different lympho-hematopoietic lineages and, therefore, again by default, exhibit multilineage repopulation potential, which would be considered by the FDA as a "clinical claim". The simultaneous detection of multiple lineages, including the T-cells and possibly B-cells, in addition to stem cells, also includes the ability of the stem cells to produce a "balanced" rather than a skewed (as shown in Table I) multilineage reconstitution. This could also be considered as a "clinical claim". Finally, the CFC-GEMM represents the most mature in vitro stem cell population. The latter may be considered as being responsible, in part, for short-term reconstitution. If a more primitive stem cell population could be detected within the final processed product, then it would be possible to argue that long-term repopulating stem cells are also present and therefore the "clinical claim" could be advocated that the test could detect cells responsible for both short- and long-term reconstitution.

It is clear that as an in vitro testing platform, HALO provides many attributes. It can be used to assess sample quality of any hematopoietic tissue used for transplantation at different levels, from "in process control" to clinical diagnostic. It has the ability to be standardized and therefore users can be subjected to proficiency testing. As an in vitro system, samples taken for testing will not compromise the patient in any way. Indeed, the opposite is the case since it would provide added safety for the patient. Because of the ability to obtain standardize results that can be used to compare intra- and inter-laboratory variations, it has the potential of being used as a "gold standard" to compare existing and new methodologies introduced into the cell processing laboratory.

A picture of the 4-plate kit is shown in FIG. 4. The following results demonstrate how this stage of development was achieved.

The HALO platform is based on a non-subjective luminescence output rather than subjective manual enumeration. Two types ofluminometers have been used. The original instrument was a relatively inexpensive TECAN GENios plate reader with Magellan software, capable of measuring absorbance, fluorescence and luminescence. A more recent acquisition has been a dedicated Molecular Devices Lmax luminometer with Softmax Pro software. The luminescence assay is a two step process by first releasing intracellular ATP and then measuring it according to the following equation:

Luciferase ATP + Luciferin + O 2 D Oxyluciferin + AMP + PPJ; + CO 2 + LIGHT

Mg 2+

The first step is to release ATP from the growing cells using an ATP releasing reagent (ATP-RR). After mixing the cultures with 20μl of this reagent followed by an incubation time of 15 min at 23 °C, 1000 of an ATP monitoring reagent (ATP-MR) is added. Addition and mixing of the reagents was performed using electronic multichannel pipettes and for most kit users, this will be the preferred and most economical method. Manual addition and mixing of reagents has been replaced by a Beckman Coulter BioMek 2000 liquid handler. This has not only allowed high-throughput processing of the 96-well plates, but a drastic reduction in well-to-well variability. The original assay system used an ATP-MR that required an immediate luminescence reading. This has now been replaced by a reagent that allows a reading to be completed during the first 30 min without significant decay. The effect of the 2 different reagents is shown in FIG. 5. Whereas the GENios instrument measures "glow type" luminescence and requires manually setting various parameters, including the gain, the Lmax can measure both "flash" and "glow" type luminescence and does not require any parameter settings. However, no two machines are alike and no standardization exists in luminescence measurements. For this reason, the term relative luminescence unit or RLU is used. For both internal studies and the kit, an ATP standard dose response is performed prior to sample measurement. This has 3 functions. First, it ensures that the luminometer is functioning correctly. Second, it ensures that the luminescence reagents are working well and have not deteriorated. Finally, it allows conversion of non-standardized RLU to a standardized ATP unit (in μM), so that results within and between laboratories can be compared over time. In short, HALO is a standardized colony-forming assay. FIG 6 shows a typical ATP standard dose response performed prior to every study. The ATP standard, ATP-RR and ATP-MR are on the right side of the kit (FIG. 4). The classical CFA procedure is shown in FIG. 3. Suffice is to say that all components have to be added separately to the tube, mixed and then dispensed into Petri dishes. Adding components individually, usually in different volumes, increases the variation of the assay due to pipetting errors, however small they might be. To reduce the variation caused by these and other errors, HALO has been designed so that only 3 culture component mixes plus the cell suspension are added together in specific ratios to form a master mix. The first mix contains fetal bovine serum and serum supplements. The second contains methyl cellulose and the third contains the growth factor mixes to stimulate different lympho-hematopoietic cell populations. Large quantities of each of the 3 mixes are prepared, aliquoted and frozen so that they can be used in an off-the-shelf manner. Once the master mix has been prepared

for a specific number of wells, 1000 is transferred to each of the wells using a manual or electronic repeater syringe. All of the culture reagents, except the cells, are provided on the left side of the HALO Kit (FIG. 4). The white 96-well plates for luminescence have a transparent bottom so that, if required, the cells can be observed under an inverted microscope. This was important during the validation of the assay, and will continue to be required during the validation procedure described in the next section.

One of the first observations made in adapting the culture conditions to a luminescence read-out, was that counting colonies in the traditional manner, i.e. after the normal incubation time and when the colonies were mature, did not correlate with luminescence. The reason was because the luminescence assay measures cell proliferation and in mature colonies, cell proliferation has either ceased or continues to occur in small areas within a colony (see below) at a low level. It was therefore necessary to define the time point or range during culture at which cell proliferation was greatest. This was determined by a time-course study in which cultures were terminated every day over a 13 day period. Before measuring luminescence, the plates were manually counted. It can be appreciated that during the very early part of the culture, no colonies are formed. However, clusters of cells can be seen and these clusters represent proliferating cell aggregates. These clusters are therefore called "proliferation units" (PU). A PU is arbitrarily defined as a cluster of 8 or more cells. However, even here care was needed, because with time, PUs become colonies and in many cases PUs can be identified within colonies. Examination of colonies shows an accumulation of cells or areas within a colony that allowed the colony to grow from a completely round ball of cells into an irregular form. The areas of irregularity were therefore considered PUs and were counted. A colony was therefore not counted individually, but rather the number of PUs within a colony was counted. Adopting this system allowed a correlation to be obtained between manual enumeration and luminescence regardless of the type of culture vessel being used.

FIGS. 7 A and B show the time course of multiple human bone marrow lympho- hematopoietic colony-forming cell populations. The dotted lines indicate manual enumeration of PUs, while the solid lines represent the luminescence readout performed after manual enumeration had been completed. For all cell populations measured, there is a parallelism between manual enumeration and luminescence, implying that at any point in time, luminescence can be used instead of a manual readout. The same relationship between readout methods exists for non-human primate, dog, rat and mouse (data not shown) and has therefore allowed multispecies comparison studies (see below).

Aii important aspect ef the previous diagrams is the exponential increase in luminescence and PUs followed by a decrease. The increase is due to a continuous increase in proliferation. At about day 10 of growth, proliferation stops or decreases rapidly and differentiation becomes the primary process to produce the colonies that can be identified under the microscope. From these growth curves, any time point on the proliferation part of the curve could be used as a luminescence readout. Day 7 has been chosen for all human stem and progenitor population endpoints, because at this time, proliferation with little or no differentiation occurs. When a cell dose response (from 2,500 to 20,000 cells per well) is performed and the luminescence measured on day 7 with the manual enumeration on days 10 and 14 are plotted against each other the correlations shown in FIGS. 8 A and B were obtained. This correlation demonstrates that the luminescence readout on day 7 can predict the manually enumerated results on days 10 and/or 14. This simple study validates the HALO procedure, thereby allowing HALO to be used in place of the CFA for most applications. It should be pointed out that HALO is not meant to replace the CFA. One is a proliferation assay, the other a differentiation assay. Each has a different readout and therefore each provides different information. Thus both assays augment each other.

A valid argument poses the question: how can it be verified that cells stimulated by single or combinations of growth factors will produce the correct types of cells if colonies cannot be identified by the morphology of the cells? An alternative phrasing of the question might be: when HALO is terminated at day 7, colonies of cells are not present, so how is it possible to ensure that the correct population is being measured? First, all cells in the culture that are capable of proliferating with or without growth factors will do so. Cells cultured in the absence of growth factors will show a response similar to the background depicted in FIGS. 7A and B. To verify that the lineages being detected by HALO contain appropriate lineage-specific cells, human bone marrow cells grown under the same conditions used for both HALO and the CFA were immunophenotyped. Multipotential stem cells were stimulated with erythropoietin (EPO), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), Interleukins 3 and 6 (IL-3 and IL-6) and stem cell factor (SCF); BFU-E were stimulated with EPO, IL-3 and SCF; GM-CFC were stimulated with GM-CSF, IL-3 and SCF, while M-CFC were stimulated with macrophage colony-stimulating factor (M-CSF). Mk-CFC were stimulated with thrombopoietin (TPO), IL-3 and IL-6, while T-CFC were stimulated with interleukin-2 (IL-2) and B-CFC were stimulated with mterleukin-7 (IL-7). After 13 days, methyl cellulose- containing cultures were diluted by the addition of PBS, and cells were transferred to 5ml

tubes, and harvested by centrifugation. After discarding the supernatants, cells were resuspended in 500/xl of phosphate buffered saline (PBS), and 100111 aliquots analyzed by flow cytometry. All cells were analyzed by gating through the pan-leukocyte CD45 marker, that is, CD45 was common to all flow cytometry panels. The results in FIG. 9A show the percentage of cells expressing this marker. It is obvious from these results that cells are present that have not been identified by the specific markers used, because the percentages do not add up to 100%. However, cells stimulated with growth factors that would produce colonies derived from CFC-GEMM contain small numbers of CD34 + , CDl 17 + and CD133 + stem cells, but 25% glycophorin-A+ erythroid cells, 9.3% monocytes (CD14), 1.6% neutrophils (CD15 + ) and 9.2% megakaryocytes (CD41 + /CD61 + ). This is indeed what would be expected since, by definition, CFC-GEMM are supposed to produce granulocytes, erythroid cells, macrophages and megakaryocytes. The BFU-E population is stimulated not only with EPO, but also SCF and IL-3. The latter is known to induce cells into the myelomonocytic lineage and by itself will stimulate the production of basophils. However, the highest proportion of cells are the glycophorin-A+ erythroid cells. The presence of megakaryocytes stimulated with high doses of EPO has been known for some time and is to be expected since EPO shares common genetic sequences with TPO. The presence of low numbers of CD34 + cells is also to be expected, since BFU-E, GM-CFC and Mk-CFC are usually all stimulated in the presence of growth factors that induce proliferation of stem cells. It is clear that those cells that should be present are indeed present. Therefore, the assumption can be made that if the same growth factors at the same concentrations are used for HALO and the CFA, then the same populations should be obtained regardless of whether they are manually identified or not. This is one of the few assumptions made in performing the HALO procedure, but this assumption is believed to be valid. FIG. 9 shows a similar analysis for the T- and B-lymphocytes stimulated by different mitogens such as phytohemaglutinin (PHA), pokeweed mitogen (PWM) and interleukin 15 (IL- 1,5). In .this case 2 panels of antibodies were used, namely CD3 (total T-cells), CD4 (T helper cells) and CD8 (T-cytotoxic cells) and CD3, CD56 (natural killer cells) and CD 19 (B- cells). The effect of activating the T-cells with different mitogens is significantly different from the growth factors alone. Furthermore, no single growth factor (IL-2, IL-7 or IL- 15) produces a single cell population; there are always other populations present. Nevertheless, these represent conditions that are used not only in the colony-forming assays, but also in immunocytotoxity assays to determine the presence of different lymphocyte populations. Measurement of such populations either as an "in process control" or after transplantation

and during patient monitoring could be extremely helpful for GvHD or graft versus leukemia (GvL) effects.

FIGS. 7 A and 7B demonstrate that HALO can be used to detect and measure multiple lineages simultaneously, hi addition to these 9 cell populations, the HALO Kit has been designed so that several other populations can be measured. Besides basic CFC-GEMM, which is designated CFC-GEMM-I, by adding TPO in addition to EPO, GM-CSF, G-CSF, IL-3, IL-6 and SCF, a CFC-GEMM-2 population has been added. By incorporating Flt3- ligand to the 7 growth factor combination, a third subset designated CFC-GEMM-3 has been included. With the addition of F13-ligand, a slightly more primitive stem cell populations is stimulated than either CFC-GEMMl or 2. There are 2 more primitive stem cell populations that are being developed for HALO. The most primitive is the colony-forming cell ~ blast (CFC-blast), followed by the high proliferation potential colony-forming cells (HPP-CFC). It is intended that the kit developed for stem cell transplantation quality control include 4-5 progenitor cell populations and 2 stem cell populations. At least 1 of the stem cell populations measured will be CFC-GEMM-2 implying short-term repopulation, while the other stem cell population would be a more primitive population, implying long-term repopulation. The other stem cell population would be either CFC-GEMM-3 or HPP-CFC. If these were detected using the conventional CFA technique, the latter population would require 14-28 days before enumeration could occur. Using HALO, CFC-GEMM-3 population is measured at the same time as the other multipotential stern cell populations. However, HPP-CFC is a more primitive population and would therefore be particularly useful to predict long-term reconstitution. The other stem cell population would be either CFC-GEMM-3 or HPP-CFC. If these were detected using the conventional CFA technique, the latter population would require 14-28 days before enumeration could occur. Using

HALO, CFC-GEMM-3 population is measured at the same time as the other multipotential stern cell populations. However, HPP-CFC is a more primitive population and would therefore be particularly useful to predict long-term reconstitution.

The majority of work performed using the HALO Platform has been to examine the toxicity of compounds on stem and progenitor cells of the blood-forming system using human as well as animal bone marrow target cells. FIGS. 10 and 11 show examples of this for doxorubicin and 5-fluorouracil, 2 commonly used anti-cancer drugs. In this particular case, HALO was compared with the CFA for 4 different cell populations so that luminescence results are shown as solid lines and clusters (PUs) are shown as dotted lines. Results similar to those in FIG. 7 were obtained, such that the results for each readout, parallel each other. FIG. 13 is a species comparison using human, dog and mouse bone marrow CFC-GEMM to examine and rank vinblastine and paclitaxel according to their toxicity. The red line shows the dose response, while the blue line is the 4-parameter logistic curve fit used to estimate the IC50 or IC90 which can be used to rank compounds in order of their toxicity. Finally, FIG. 14 is a species comparison using a single compound (C 1) tested on CFC-GEMM and GM-CFC from human, dog and mouse bone marrow. In addition, a comparison between HALO and the CFA was also performed for all parameters. The results demonstrate the conversion of non-standardized RLU values to standardized ATP (μM) values. In addition, from the 4-parameter logistic curves, both an IC50 and IC90 were estimated. More important however, was the comparison of the HALO and CFA results. A complete dose response could be obtained with respect to the proliferative status of the populations in response to the compound using HALO. However, a :respectable dose response curve was only obtained for canine bone marrow CFC-GEMM and GM-CFC mature colonies. For both human and mouse, no differentiation into colonies was observed. This implies that the compound allowed proliferation, but blocked differentiation. This illustrates that if the CFA were performed alone, the interpretation of the results would be different. However, it also emphasizes the fact that in some cases, both HALO and CFA may be required to complete the story. As stated above, HALO is not meant to replace the CFA. It can, however, provide a more rapid, multiparameter result. For the present application, it is possible that upon stem cell transplantation, the cells may engraft and grow and multilineage reconstitution may occur, but mature blood elements may be lacking in the periphery. If this were the case, one possibility might be that therapeutic agents caused a block in one or more lineages, which could be confirmed by the CFA technique.

Quality control for these and all future batches will be performed as follows. Random samples of each of the kit reagents from the same batch will be sent from Bioserv. Fresh, whole normal and mobilized peripheral blood, bone marrow and umbilical cord blood will be obtained from a vendor and the mononuclear cell (MNC) fraction prepared by density gradient centrifugation using Ficoll-Paque Plus (Amersham, Inc) in Lymphosep (Greiner BioOne, Inc) tubes. These tubes contain a porous frit upon which the Ficoll is added. The tubes are centrifuged to force the Ficoll to the bottom of the tube. The whole tissue is diluted with phosphate buffered saline (PBS) and then poured onto the fit and the tube centrifuged at 1000 x g for 10 min. Most of the plasma is removed and the MNCs poured into another tube and washed with PBS. A nucleated cell count, CD34 and viability (7-aminoactinomycin-D) analysis by flow cytometry is performed on the whole tissue prior to separation and on the washed, separated MNC fraction. After adjusting the cell concentration of each of the target cell preparations, the cells will be added to each of the pre-made and pre-mixed tubes for the cell populations to be measured. After mixing the cells with the tissue culture reagents, 1 OOμ.1 will be dispensed using a Hamilton AT Plus 2 liquid handler into each of the wells of a 96-well luminometer plate. Since the tissue culture master mixes (pre-mixed reagents plus target cells) contain viscous methyl cellulose, they cannot be dispensed using normal pipette tips. A positive displacement pipette or repeater syringe has to be used. For research purposes a repeater syringe is normally employed. Since quality control is a routine procedure, this can be performed automatically using a liquid handler. The only liquid handler that uses positive displacement tips is believed to be the Hamilton AT Plus 2 instrument. Incubation and luminescence measurement is performed as described. The results as a range of values for each cell population measured for a specific batch of reagents, will be provided as an addendum to the HALO Kit manual. This provides users of the kit and indication of expected values and therefore information on interpreting the results obtained.

The results demonstrate how the HALO Platform has been validated and is used to analyze the effects of compounds on hematopoietic cells. Three different stem cell populations are already available for stem cell testing and another 2 will become available shortly. In addition, 9 different populations from each of the committed lympho- hematopoietic lineages can be detected and measured. Therefore, if required, 14 different populations could be analyzed from peripheral blood,, bone marrow and/or umbilical cord blood. HALO is a sophisticated, rapid, robust and reliable test system well suited for the "in process control" application described below and future in vitro clinical diagnostic applications for which this proposal will lay the important groundwork.

EXAMPLE 2: A modified HALO Kit for "in process control" with the ability to determine engraftment and multilineage reconstitution potential

This aim will be achieved in two stages. The first stage is to determine the type and number of cell populations that will be detected and measured. The second stage involves mixing and incorporating the reagents for these populations into a kit that can be used as easily, rapidly and efficiently as possible.

If tissues are processed so that hematopoietic stem cells are transplanted, then it follows that the in vitro GM-CFC progenitor cell is not the cell population of choice for stem cell quality or engraftment, but rather the in vitro multipotential stem cell population or CFC- GEMM. Since the CFC-GEMM population is the most mature in vitro stem cell population that can be detected and contains CD34 + , CD 117+ and CD 133+ stem cell (FIG. 9A), it is also useful to predict short-term engraftment. The in vitro multipotential stem cell population, CFC-GEMM, is a prime candidate for this since its presence and growth potential can be detected and measured in 7 days using HALO. Committed progenitor cell populations are also detected' after 7 days in culture. Therefore the CFC-GEMM would be one of 2 stem cell population incorporated into the kit.

Hematopoietic commitment into the erythropoietic, myelomonocytic and megakaryocytic pathways can be detected by the presence of the first committed descendants of the CFC-GEMM population, namely the GM-CFC, BFU-E, Mk-CFC progenitor cell populations. By definition, the CFC-GEMM population gives rise to cells in these hematopoietic lineages, and therefore provides an indication as to whether the quality of the stem cells allows production of lineage-specific cells. That balanced and not lopsided commitment occurs would be determined by the presence of the GM-CFC, BFU-E and Mk- CFC. If one or more of these populations are missing (as seen in Table 1), then growth factor therapy by the physician might be considered to achieve a more balanced reconstitution.

As demonstrated in FIG. 9 A, stimulation of CFC-GEMM does not produce either T- cells or B-cells. The reason for this is seen in FIG. 1. The lymphoid lineages digress from the primary hematopoietic lineages early in the stem cell hierarchy. The CFC-blast population is considered the "fork-in-road" with respect to lymphopoiesis and hematopoiesis. Reconstitution of the T-cell lineage occurs- at a much later time point than the hematopoietic lineages. For several years, T-cell depletion of the transplanted cells has been considered important from two opposing aspects, GvHD and GvL. The ability of the transplanted stem cells to produce T-cells is therefore of importance. This ability can be tested by the inclusion of the T-CFC population with the other committed hematopoietic progenitor cell populations.

Since the formation of lymphopoietic cells is dependent on the presence of primitive cells, it follows that the inclusion of a more primitive stem cell population than the CFC- GEMM, would be beneficial to the predictive value of the kit. However, the second stem cell population is more problematical for several reasons. On the one hand, the more primitive the stem cell, the greater the quiescent state, i.e. fewer cells in cycle. On the other hand, when stimulated, the more primitive the stem cell, the greater the proliferative potential and the larger the colony, but the longer it takes to be detected. However, detecting such a primitive stem cell population would predict that long-term repopulation cells are present in the transplant. Prior to finalizing the make-up of the kit, a decision has to be made regarding whether a second stem cell population can be included and if so, which population it will be and how long it will take to be detected using HALO technology.

The growth factor Flt3-ligand has been shown to expand and activate NK cells as well as dendritic cells and other T-cell populations. As mentioned above, one of the stem cell populations that can be measured with the HALO kit is designated CFC-GEMM 3, which is stimulated with EPO, GM-CSF, G-CSF, IL-3, IL-6, SCF, TPO and Flt3-ligand. Changing the growth factor combination to include IL-3, IL-6, G-CSF, GM-CSF SCF and Flt3-ligand has been shown to induce the equivalent of the murine HPP-CFC cell population for human cells. Originally discovered in 1979 by Bradley and Hodgson, the high proliferative potential colony-forming cell (HPP-CFC) has been used as a marker for primitive stem cells, both in basic and clinical research. The human equivalent was first detected in 1989 by McNiece et al, who used high doses of GM -1 CSF and IL-3 to produce these colonies. The hallmark of this population is the production of macroscopic colonies, several millimeters in diameter that take between 14 and 21 days to form. This population is also relatively insensitive or resistant to 5-fluorouracil (5-FU) indicating that it is not normally in cell cycle. The CFC-blast population would be a relatively easy population to detect if it were not for the fact that to verify this cell, primary colonies have to be removed and replated a second time. The long-term culture-initiating cell or LTC-IC, also called the cobblestone area-forming cell (CAFC), is the most primitive in vitro stem cell.population that can be detected, but requires a large number of procedures and entails a complicated process requiring 5-7 weeks to perform. From a practical point of view, neither of these stem cell populations would be applicable to a kit format or for clinical purposes.

This leaves the CFC-GEMM-3 and HPP-CFC populations for possible kit inclusion. However, these two populations may be one and the same. Many of the growth factors required are similar for both populations and the differentiating factor might be the time

required for measurement. Nevertheless, one of the requirements for this population is to measure both hematopoietic and lymphopoietic cells and to this end, combinations of growth factors would be used that would stimulate both systems. To decide which population will most likely be included into the new kit, the following studies will be performed. (a) Growth factor combinations'. Defining a specific growth factor combination is not an easy task because the combination that may be used for the present studies may change in the future. It is not only necessary to be able to stimulate primitive stem cells to produce multiple lineages, but also to accomplish this in a reasonable time period. If the results require a slightly longer incubation period than that of the other populations measured that would correlate with long-term reconstitution and lymphocyte repopulation, then this would be a small price to pay for this important information. Rather than studying multiple growth factor combinations, 2-3 will be focused upon with the goal of establishing an in vitro stem cell population that can produce both lympho- and hematopoietic lineages and exhibit the potential for long-term repopulation. One growth factor combination would include GM- CSF, G-CSF, SCF, IL-3, IL-6, TPO and Flt-3 ligand, while another would use the same combination, but with IL-2. If results do not produce the required endpoints, then other additional growth factors, such as IL-I, IL-4, IL-7 and IL- 15 might be added.

For all studies, the following basic protocol will be used. Mononuclear cells from target tissues will be prepared using Leucosep tubes (Greiner Bio-One). These 15ml or 50ml have a porous filter disc or flit, which allows easy, rapid and above all, standardized separation of MNCs using a density gradient medium such as Ficoll-Hypaque Plus (Pharmacia) or Nycoprep (Greiner Bio-One). After washing and resuspension in medium, the nucleated cell count will be determined and an aliquot used for phenotypic analysis as described in (b) below. The cell concentration will be adjusted for culture and added to premixed tubes containing the serum mix, methyl cellulose mix and growth factor mix to be tested, to produce a master mix. This will then be divided into wells of a 96-well plate containing 104μl of the master mix. Since one of the aims is to test whether colonies containing all 8 lineages are obtained, a measure of proliferation by luminescence will be performed only after full growth of the colonies as been attained. Furthermore, since the cell composition of the colonies is also required, sufficient cell replicates will be used to determine both proliferative status and phenotypic analysis. In order to obtain sufficient cells for phenotypic analysis, 8-12 replicates will be prepared specifically for this purpose and another 8-12 replicates to measure proliferative status by luminescence. This means that 16- 24 wells will be available for manual enumeration of the colonies.

(b) Phenotypic analysis: The results from studies shown in FIGS. 9A and 9B were performed by adding PBS to each well and mixing to reduce the viscosity of the methyl cellulose. This was repeated several times so that the cells could eventually be centrifuged and resuspended in PBS .and incubated with fluorochrome-'conjugated antibodies to different surface markers. To perform several phenotypic panels, all of the cells were pooled and then aliquoted into different tubes. Each of the tubes containing different antibody panels were analyzed using an EPIC XL/MCL flow cytometer. With large numbers of wells, this is a tedious and time-consuming process. This and other techniques will be significantly improved by the acquisition in the second half of 2004 of a Beckman Coulter FX500 flow cytometer capable of using a 96-well plate formation. Depending on the number of antibody panels required, the phenotypic composition of cells present in each well will be analyzed. If 12 replicate wells are available and 6 antibody panels are to be measured, each panel can be performed in duplicate and the whole process, semi-automated. Using a liquid handler, PBS will be added and the well contents mixed. After centrifugation of the plates, the antibody cocktails will be added and after a lOmin incubation period, ImmunoPrep reagents (Beckman Coulter) which lyse red blood cells, stabilize and fix the remaining cell suspension will also be added. The phenotypic composition of the cells in each well will then be acquired and analyzed. All panels used will contain the CD45 pan-leukocyte membrane marker. Phenotypic analysis will include antibodies to detect the presence of those markers shown in FIGS. 9 A and 9B, as well as CD90 (Thy 1 antigen present on primitive stem cells), HLA-DR (histocompatability antigen) and CD38 (progenitor cell lineage marker). Panels will consist of 4 antibodies and the absolute number of each cell population will be obtained using beads which allow conversion to a concentration per μ\ of sample. The information obtained from phenotypic analysis must show that cells from all 8 lineages are present. This, together with manual and luminescence enumeration will allow a decision to be made on the growth factor combination required for inclusion into the final kit. However, to determine the optimal time to measure luminescence and to ensure that the stem cell population is sufficiently primitive to determine long-term repopulation potential, 2 other studies have to be performed.

(c) Time course study; For the chosen growth factor combination, a similar time course study to that shown in FIG. 7 will be necessary. The aim is to determine the optimal time to measure luminescence. For this, cultures in 96-well plates will be prepared so that 1 plate can be terminated on each day of the incubation period. Sufficient replicates will also be prepared so that both luminescence and phenotypic analysis can be performed. On each day, the number of PUs and colonies (if present) will be counted manually. Half of the

replicates will then be subjected to luminescence measurement while the other half will undergo phenotypic analysis. There are 3 possible types of growth curve. The first would be similar that that obtained in FIG. 7A in which exponential proliferation followed by a decrease would be observed. The second might be a continuous proliferation as seen for many of the populations in FIG. 7B. The third might be a biphasic proliferation curve in which the first phase would be an initial proliferation phase, followed by a second, but greater proliferation phase that would produce the large numbers of cells required to make up a macroscopic colony. In the former case, a time point would have to be used that would ensure the presence of early non-committed cells, while in the second case, this would be later. If the third case were to occur, then the optimal time to measure luminescence would be during this second phase of proliferation.

(d) Secondary plating: Long-term repopulation requires that primitive stem cells are present and, by definition, the stem cells exhibit extensive self renewal potential. This means that if stem cells with different degrees of "sternness" are present, the more mature cells would exhibit limited self-renewal capacity, while the more primitive would exhibit greater self renewal capacity. To ensure that cells are present with greater self-renewal capacity, replicate cultures will be prepared and at a time at which proliferation is optimal and/or maximum (determined by (c)), the cells from individual wells will be removed and replated into secondary wells under the same conditions. The growth of secondary cultures and the presence of phenotypically positive stem cells, implies that primitive stem cells were present in the initial culture and that long-term repopulation potential can be predicted. This, together with the other studies in this aim, would define the growth factor combination to be used for the population being measured, and the optimal time for luminescence measurement. Once the more primitive stem cell population (designated stem cell-1) has been defined in the above studies, the new kit can be designed. There are 2 possible configurations for a kit of this kind to be used in a potential clinical situation. The first would be similar to the HALO research kit shown in FIG. 4. The left side of the kit contains the 3 mixes required to culture the cells. The latter are provided by the user. The right side of the kit contains the ATP standard and the reagents for measuring luminescence. The growth factor combinations (shown in FIG. 4 as separate vials), in this case, for each of 4 populations to be measured, are added in the required ratios with the cells, mixed together and the master mix dispensed into the wells using a repeater syringe. Although this is an easy procedure, there is an even easier and more rapid way of performing this task, which has been alluded to (a) above. All of the reagents for each of the cell populations to be measured would be provided in pre-made, pre-

mixed individual tubes. The user only has to add the cells, mix and dispense the master mix into the wells. This is more efficient, saves time and is even more robust and reliable since the amount of pipetting needed is reduced to a bare minimum. There would, however, be 2 variations on this theme determined by the number of replicates used and populations tested. FIG. 12 shows these 2 variations. In variation 1, 8 replicates for each population are prepared and plated in columns from A to H. If 6 populations (Stem cell 1, CFC-GEMM-2, BFU-E, GM-CFC, MK-CFC and T-CFC) are to be detected, 2 samples can be accommodated on each plate. In the second variation, 6 replicates are plated in rows from 1-6 and 7-12. However, in this case, 8 populations from 2 samples can be accommodated on 1 , 96-well plate. Besides the 6 populations given above, the B-cell progenitor, B-CFC stimulated with IL-7, could added, and a background could also be included to which no growth factors are added. The background is an important control, since it measures the ability of cells to spontaneously proliferate in the absence of growth factors and provides a value above which all other populations should exhibit luminescence. By including the background, it is then possible to arbitrarily define the state of proliferation. For example, if the RLU or ATP values for a population are greater than 2-3 times the standard deviation of the background, proliferation can be said to have occurred. An extra, non-sterile plate would be provided in the kit to perform the ATP standard dose response. Values for each population from each tissue would be provided to the user in the manual as a historical range that would be obtained, in part, from the results as derived.

Base line data each of normal and mobilized peripheral blood, bone marrow and umbilical cord blood using the new version of the HALO Kit will be obtained and the results will be compared with those obtained at the same time using the classical colony-forming assay. The primary goal of this aim is to validate the newly developed HALO Kit against the conventional colony-forming assay procedure. The secondary goal is to accumulate base-line data for normal tissue. The third goal is that once validation of the HALO Kit has been performed, it can then be used for further studies. To a certain extent, the outcome of this aim is already known from FIGS. 8 A and 8B. However, these results were only obtained with one tissue type and a limited number of samples. To achieve this aim, a minimum of 20 samples of normal and mobilized peripheral blood, bone marrow and umbilical cord blood will each be assessed. The tissues will be obtained from a commercial vendor, e.g. Cambrex/Poietics BioSciences, Walkersville, MD. The power statistic of this number of samples is 0.95 if the alpha value is 0.05 (PO.05). If more samples are available, these will

also be analyzed. The data obtained will be included as normal historical values in the manual that will accompany the new kit.

There are, in fact, at least 5 alternatives to performing this part of the proposal. The different alternatives are dependent on how the classical colony- forming assay is carried out. The HALO version defined above would remain the same throughout.

Alternative 1 : HALO is performed as described above using whatever version is deemed suitable. The CFA would be carried out in a traditional manner (FIG. 3A), by adding all the reagents, including the growth factors separately so that the total volume would be sufficient for 2-3 replicates of 1 ml each (total volume prepared 3.5-4ml). Standard 1 ml Petri dishes would be used. After incubation, the cluster/colony count would be evaluated manually for the whole plate. The disadvantages to this technique is that large quantities of culture reagents are used, which also increases the cost of each assay, and that setting up the assays would take considerable time, especially if large numbers of samples are to be analyzed at the same time. Alternative 2: The second alternative is to use ready-made reagents provided by Stem

Cell Technologies (Vancouver, BC), instead of adding the reagents separately. The problem with this is that reagents for each of the cell populations to be tested, namely stem cell-1, CFC-GEMM-2, GM-CFC, BFU-E, Mk-CFC, T-CFC and possibly B-CFC are not all available in the same format, so that most cell populations would have to be prepared as in the first alternative.

Alternative 3: Li this case each CFA assay would be prepared for quadruplicate cultures of 1 OOl 11 each (total culture volume of 600 ml) as shown in FIG. 3B. The plates used in this micro-CFA procedure and previously developed are 1 ml Petri dishes which have 4 wells each with a surface area of 95.03 mm . The clusters/colonies are counted in each well. Although the quantities are significantly reduced in this microculture method and would be the same volume/culture as that for HALO, the time to set-up the assay would be a limiting factor if many samples are obtained on the same day.

Alternative 4: The fourth alternative is similar to the third, but in this case, a single master mix is prepared so that sufficient volume is available for both the HALO and the A- replicate, micro-CFA.

Alternative 5: The last alternative is that a single master mix is prepared for each sample to be assayed on each of the cell populations, but both HALO and CFA and plated in duplicate 96-well plates using the same number of replicates. One 96-well plate would be counted for clusters prior to luminescence measurement on the designated day for HALO,

while the other plate would remain in the ineubator to produce mature colonies which would be counted before measuring luminescence. The difference in master mix volume between alternatives 4 and 5 would be between 400-500121. The difference in total volume would be 2.25ml for 16 wells and 2.0 ml for 12 wells. Despite the fact that both clusters and colonies are counted on different days, it usually takes between 1-1.5h to manually enumerate a 96- well plate, whereas it takes 3-5 times longer for Petri dishes. More important, however, is that the samples will all be handled in the same manner, for both types of assay, thereby reducing variability between the assays and increasing the significance between the assays. This is considered the best alternative because more comparable information can be obtained. Indeed, 4 parameters can be measured. These are manually enumerated cluster or PU number versus proliferation by luminescence and manual enumeration of differentiated cluster/colony formation versus proliferation by luminescence at the mature colony time point. The ratio of mature colony numbers to luminescence values provides a direct indication of the amount of proliferation occurring during the differentiation phase. An analysis of variance (ANOVA) will allow correlation and statistical differences between the different parameter measurements to be ascertained. The prediction would be that similar correlation curves to those shown in FIGS. 8A and B would be obtained for each population and tissue tested. Thus, the necessary validation would be obtained to allow HALO to be used in place of the classical CFA. In addition, this second aim will provide the baseline values for normal tissues required for future clinical trial studies.

EXAMPLE 3: Use of the HALO Kit to test clinical samples

The 2 aims of this example are: (1) a HALO Kit to retrospectively test "blind", patient clinical samples to predict engraftment and multilineage potential and to compare the results with the patient clmieal outeome at the end of the study and (2) use of the HALO Kit to compare samples from the same patients at one or more stem cell transplantation centers.

Although the validation to use the new HALO Kit in place of the classical colony- forming assays is part of the Examples described above, further validation uses patient samples. These will be frozen samples from transplant donors. The samples will be obtained from transplant centers and tested "blind". The results will be compared with those of the transplanted patient to determine if the cells grew, engrafted and exhibited multilineage reconstitution.

Use of the HALO Kit to compare samples from the same patients at one or more stem cell transplantation centers. Kits are prepared and distributed to one or more transplantation centers. Samples from the same patient would then be analyzed at all testing centers and the

results compared. The goal is to demonstrate that the HALO Kit functions the same at all testing centers and that the results obtained are within acceptable ranges for each of the populations under study. This aim will pre-empt a larger clinical study that would be required as a basis for potential "clinical claims" that will be submitted to the FDA for review.

The newly developed HALO Kit will be used to retrospectively test "blind", patient clinical samples to predict engraftment and multilineage potential and to compare the results with the patient clinical outcome at the end of the study.

This clinical study will not involve fresh "in process" quality control of tissue that is destined to be transplanted. The tissue to be used for this Example will be frozen, processed samples that have, at some previous time point, been infused into a patient and for whom the clinical outcome is known. The clinical outcome will not be divulged during the course of the study, but at the end, when all the in vitro HALO data have been accumulated and analyzed. Thus, HALO will be performed "blind". The only information is the origin of the original tissue, that is, normal or mobilized peripheral blood, bone marrow or cord blood and whether the tissue had been processed to the MNC stage or the CD34 + stage, since this will depend on the number of cells plated in vitro.

A minimum of 20 frozen patient/donor samples each of mobilized peripheral blood, bone marrow and umbilical cord blood will be tested. This number provides the power statistic which would allow 95% confidence limits to be obtained. However, if time and funds permit, as many samples as possible will be processed in order to achieve better prediction statistics.

Preparation and the HALO procedure is described below.

(a) Sample thawing: Each frozen sample will be carefully thawed to avoid undue cell death and the release of DNA which can form clumps in the cell suspension and entrap viable cells, thereby reducing the nucleated cell count. If the cells have been frozen using dimethylsulphoxide (DMSO) as cryopreservant, one of two thawing methods is available: The first is to rapidly thaw the cells at 37 0 C and transfer the cells to PBS containing heparin and DNase. The second method also involves rapidly thawing the cells, but then all cells from the vial are transferred to an empty tube and a mixture containing PBS and serum at different volumes is added over a 10 min period. This timed DMSO dilution protocol is not usually the method used when frozen cells are transplanted. Normally the cells are rapidly thawed and infused almost immediately, although some toxicity from DMSO has been seen. It is possible that the presence of serum also results in clumping of the cells. An alternative

protocol would be to perform a timed DMSO dilution procedure, but instead of using serum, heparin and DNase are used instead. Regardless of which procedure is used, the presence of DMSO is toxic in culture and its concentration must be reduced so that any cell growth is not masked. Cord blood cells are usually frozen in Hetastarch (HES) and thawed quickly at 37°C. As discussed in the above Examples, a SOP for sample thawing will be ascertained during these studies and might be different for each of the different tissues tested. This SOP will also help in the above Examples, where different transplantation centers will be performing the same procedures.

(b) Pre-culture methodology: Once the cells have been thawed, washed and resuspended in 300-5000 of medium (Iscove's Modified Dulbecco's Medium, IMDM), a nucleated cell count will be performed using a Beckman Coulter Z2 particle counter. A sample of the cell suspension will then be analyzed by flow cytometry for viability using 7- AAD as well as the presence of stem cell subsets and the primary lineage-specific populations. The stem cell subsets will include CD34, CD 117, CD 133 and CD90. Content of CD34 + cells will be performed using the Beckman Coulter Stem Cell Kit and analyzed using the ISHAGE protocol. This relies on all CD34 + cells being CD45 + . If the sample has been designated to contain CD34 + cells, the CD34 analysis will determine whether the sample contains only CD34 + cells or whether other CD45 + /CD34 + cells are present. If the latter is the case, then other antigens will be determined. These will include glycophorin-A (erythroid cells), CD 14 (monocytes), CD 15 (neutrophils), CD41 ICD61 (megakaryocytes), the T-cell markers, CD3, CD4 and CD8 and the NK and B-cell markers CD56 and CD 19 respectively. All antibodies will be used in panels of 4, one of which will be the pan- leukocyte antibody to CD45, through which all other antibodies will be gated. Between 1 μ\ and 20μ.l of sample will be used for each panel, but if insufficient cells are available for culture, then the number of panels will be reduced and priority given to using cells for

HALO. The reason for performing phenotypic analysis is .to ascertain the composition of the starting cell suspension, so that it can be related to populations determined by HALO.

(c) HALO: The majority of the cells suspension will be adjusted to the cell concentration required for cell culture. Normally, if only MNCs are available, then the cell concentration required would be about 0.5 x 10 6 cells/ml or about 0.5 x 10 4 cells/100/xl culture. If the starting cell suspension has been purified to CD34 + cells, the starting concentration will be reduced 10-100 fold. The appropriate cell concentration in a designated volume will then be added to each tube containing the pre-mixed cultured reagents for the cell populations to be measured according to the configuration determined above (FIG. 12).

After incubation at 37°C in a humidified atmosphere containing 5% CO 2 and 5% O 2 for the designated period of time, the plates are transferred to a 23°C humidified incubator with 5% CO 2 and left to equilibrate at that temperature for at least 30 min. Thereafter, the plates will be placed in the liquid handler and the ATP releasing reagent and monitoring reagent added and mixed with the culture components automatically. Finally the plates will be read in the Lmax luminometer and the data stored. Normally the results are exported to a text file which is then imported to an Excel spreadsheet, from where the results can be further analyzed using other software programs where necessary

(d) Data evaluation: A minimum of 20 samples from each tissue type will be analyzed. Thus a total of at least 60 samples will be processed. Since HALO analysis always involves an ATP standard dose response, all RLU values will be converted to ATP (/xM) values. Variation in the daily ATP standard dose response does occur due to use of a new batch of ATP and/or ATP-MR. In order that samples tested at different times can be compared, a normalized ATP standard curve will be produced at the end of the study and all samples normalized against this standard curve. In this way, day-to-day internal variations can be reduced and the response of the samples statistically analyzed and compared. From these data, the answers to the questions 1, 2, 3, 5, 7, and 9 for each sample will be tabulated. These predictive answers will then be compared with the clinical outcome for each patient sample by answering the questions 4, 6, 8 and 10. The hypothesis is that the in vitro data will predict the clinical outcome. If stem cells were present (Question 1) and they grew in HALO (Question 2), then engraftment should have taken place (Question 4). If Question 5 indicated that the stem cells became committed by the stimulation of CFC-GEMM, then the patient should have exhibited multilineage reconstitution (Question 6). If all lineages tested in HALO were present (Question 7), then it would be expected that a balanced or near normal multilineage reconstitution would have taken place (Question 8). If only certain progenitor cells grew in culture, then these lineages would be expected in the patient. If T-cells, were produced in culture (Question 9), then it would be expected that not only primitive stem cells would be present, but that either GvHD or GvL might have occurred (Question 10). If HALO indicated that both short- and long-term stem cells were present (Question 3), then short- and long-term engraftment should have occurred and the patient might still be alive (Question 11) barring other eventualities.

The samples used for this aim would be frozen, although if freshly processed tissue was made available, it would be incorporated into the study. The number of samples available for testing at several sites might also be a limiting factor, since sufficient samples

have to be kept for regulatory purposes. It is important that as little information about the sample and patient be known at the time of testing. Therefore, to avoid any bias, the center providing the sample would distribute the sample to other testing centers, but would not test the sample themselves. This assumes that more than 1 transplant center would be involved in the study.

In order to test the samples at all facilities, they will have to be shipped by overnight courier. The participating centers will have sufficient kit materials to perform the studies, especially if fresh samples are used.

It is imperative that the samples are treated in exactly the same manner at all facilities prior to addition to the tissue culture mixes. This means that if the samples are frozen, each facility testing the sample will thaw it and handle it according to a designated SOP to be determined prior to the start of this study by all concerned. Thawing and handling frozen material is an important first step and that developed procedures will help define a consensus SOP. Not all centers have similar instrumentation to evaluate the nucleated cell count. However, cell counting is a procedure that is performed under standardized conditions in most transplantation centers and where possible, these conditions will be used at all facilities. Standardized phenotypic analysis of CD34 + cells and their viability would be according to the ISHAGE protocol. Similarly, SOPs will ensure that the adjusted cell concentration for in vitro HALO testing is performed in the same manner and that the same cell concentration is used for a specific sample at all testing facilities.

Despite an endeavor to try and ensure that all procedures are standardized, there are some aspects which will not come under this umbrella. One of the most important is how the cells are incubated. This may appear to be a minor point, but there is in fact a significant difference regarding whether cells are incubated under atmospheric (21%) or low (5%) oxygen tension conditions. Bradley et al first demonstrate the advantageous effect of culturing GM-CFC under low oxygen. In 1982, Rich and Kubanek demonstrated that erythropoietic cells exhibited a greater plating efficiency in culture under low oxygen tension and that the inclusion of reducing agents such as beta-mercaptoethanol, alpha-thioglycerol or even vitamin E and reduced glutathione, had an additive effect when cells were grown under low oxygen. All HALO cultures are incubated under 5% oxygen tension which is approximately equivalent to about 45mmHg. This is also the venous partial oxygen tension in many organs, including the bone marrow. The increased plating efficiency, resulting from reduced oxygen toxicity caused by free radicals, allows greater sensitivity and therefore increased number of colonies in the CFA and higher luminescence values. Tissue culture

S9

incubators are now readily available to allow cells to be grown under low oxygen tension. However, it must be expected that few stem cell transplant centers will have access to these tissue culture incubators. Tissue culture of the 96-well plates for HALO will probably be performed in normal incubators gassed with 5% CO 2 . Rather than just indicating cell growth, HALO can provide more important information. The indication that cells will growth is also a prediction of engraftment. The configuration of the new HALO Kit will incorporate a detection system that will predict both short- and long-term engraftment, by allowing stem cells with different degrees of growth potential to be detected simultaneously. By including other more mature blood-forming cell populations, the kit can also be used to determine if some or all of the blood cell lineages are going to be produced in the patient.

The research HALO Kit Platform provides a more rapid, reliable, robust, non- subjective and standardized testing system with high-throughput capability than the classical colony- forming assay. In contrast to the latter, HALO also allows flexibility and versatility due to its ability to detect and measure multiple cell populations from multiple species simultaneously. Accordingly, it is believed that the HALO Kit Platform is a superior product.

EXAMPLE 4: The HALO Stem and Progenitor Cell Quality Control (SPC-QC) Platform: A replacement for the colony- forming assay for stem cell transplantation processing and cord blood storage.

The colony-forming assay has been used in basic and clinical research for nearly forty years. With the exception of recombinant growth factors and serum- free conditions, the assay has changed little. Culture under low oxygen tension was introduced by Bradley et al. in 1978 for GM-CFC and Rich et al. in 1982 for BFU-E and CFU-E. This, together with miniaturization of the culture eventually led to the development of HALO.

The drawbacks to the colony- forming assay are as follows: (a) time-consuming to perform, (b) high degree of technical expertise required, (c) lack of standardized colony enumeration procedures, (d) manual enumeration of colonies is highly subjective, (e) lineage and species comparisons are difficult to perform, (f) low throughput and (g) assays are expensive to perform.

HALO is a colony- forming proliferation assay without a microscope. The similarities between HALO and the colony-forming assays are as follows: (a) HALO is based on the colony-forming assay, (b) both are clonogenic assays, (c) both are functional assays, (d) both

use methyl cellulose to immobilize cells and (e) both use the same growth factors and cytokines.

HALO is a proliferation assay whereas colony formation is a differentiation assay. Proliferation occurs before differentiation, differentiation requires proliferation, but proliferation does not require differentiation. Under normal conditions, if proliferation occurs, differentiation will follow, i.e., proliferation will predict a differentiation response. Table 3: How HALO differs from the colony-forming assay

HALO detects proliferation as follows. As cells proliferate, intracellular ATP increases. The release of intracellular ATP after 7 days incubation drives a luciferin and luciferase reaction. Luciferase

ATP + Luciferin + O 2 D Oxyluciferin + AMP + PP 1 ; + CO 2 + LIGHT

Mg 2+

The bioluminescence is detected and measured in a plate luminometer (which is required to perform HALO). The luminometer output is non-standardized Relative Luminescence Units (RLU). RLU is converted to standardized ATP units (μM) from ATP standard curve.

FIG. 15 depicts how the non-standardized, relative luminescence unit (RLU) output of the luminometer is converted to standardized ATP units by performing an ATP dose response prior to measuring samples. The software that drives the luminometer can usually be programmed to perform these operations automatically. HALO does not correlate with traditional colony counts. The reason for this is because when colonies are produced, they contain primarily differentiated cells. Since HALO is not a differentiation assay, there is little or no correlation between individual differentiated colonies (shown in FIG. 16) and proliferation. HALO does correlate with "proliferation units" or clusters within a colony. When cells are incubated in methyl cellulose, small clusters are formed. These clusters consist of undifferentiated, proliferating cells which may be referred to as "proliferation units". Although more difficult and time- consuming to count, it is these "proliferation units" that correlate with the luminescence proliferation assay (FIG. 17). FIG. 18 shows two important aspects of HALO. First, the kinetics of luminescence as a function of the number of "proliferation units" or clusters manually enumerated as growth occurs in CFC-GEMM, BFU-E and GM-CFC cultures. The kinetics of both the number of "proliferation units" and luminescence are parallel indicating a correlation between the two parameters. Second, the graph provides information that at 7 days, luminescence can be measured with little or no differentiation. FIG. 19 shows a slightly different variation, where there is a direct correlation between luminescence and the number of "proliferation units" counted as a function of cell dose. Accordingly, HALO and manual enumeration of "proliferation units" predicts differentiation response and HALO can replace the colony-forming assay.

The sensitivity of the HALO platform with respect to cell concentration is depicted in FIG. 20. A HALO multiple population response profile using fresh human bone marrow mononuclear cells is depicted in FIG. 21.

FIGS. 22A-22D show the different configuration of the HALO SPC-QC platform. For general screening of samples for growth potential, the single stem cell population (CFC- GEMM), with or without a control would be sufficient. For more advanced analysis of the sample, the 2-stem cell or 4-population kit could be used. To analyze all blood-producing lineages, the HALO 7-population kit would be used. This would provide possible predictive information regarding short- and long-term engraftment and repopulation potential prior to transplant.

From initial studies with cord blood, two factors interfere with HALO. The first is high concentrations of whole erythrocytes (FIG. 23), which provide oxygen causing an

imbalance in the luciferin/lueiferase reaction. The second is hemoglobin, released from lysed erythrocytes. This has been known to inhibit luminescence. FIG. 24 depicts the HALO-7 population response using umbilical cord blood. The results in FIG. 25 were obtained from three cord blood samples obtained from the Puget Sound Blood Center. The results indicate that the total number of colonies manually counted show a similar pattern of results to those obtained by HALO.

Besides time and cost savings, HALO allows: (a) less skilled personnel required- does not rely on a single person, (b) time to proficiency: 1-2 weeks, (c) rapid, standardized, non-subjective and reproducible, (d) flexibility and expandability, (e) automated with high- throughput capability, (f) direct indicator of stem cell growth potential, (g) incorporation into proficiency testing and (h) may be predictive for long- and short term engraftment and reconstitution potential.

The invention is further described by the following numbered paragraphs:

1. A method for determine engraftment and multilineage reconstitution potential comprising: (a) preparing one or more cell suspensions, (b) adding the one or more cell suspensions to a pre-dispensed and pre-mixed tube comprising a serum mix, a methyl cellulose mix, a growth factor mix, a medium, an ATP-releasing reagent, and an ATP luminescence-monitoring reagent thereby forming one or more samples, (c) dispensing one or more samples into one or more vessels, (d) incubating for 7 days at 37 C in a humidified atmosphere with 5% CO 2 and 5% O 2 , (e) performing an ATP standard dose response and measuring luminescence, (f) measuring relative luminescence of the one or more samples, (g) conversion of relative luminescence units of the one or more samples to standardized ATP units and (h) correlating the standardized ATP units to engraftment and multilineage reconstitution potential. 2. The method of paragraph 1 wherein the cell suspension comprises colony- forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM).

3. The method of any one of paragraphs 1-2 wherein the cell suspension comprises high proliferative potential stem and progenitor cell (HPP-SP).

4. The method of any one of paragraphs 1-3 wherein the cell suspension comprises burst-forming unit erythroid (BFU-E).

5. The method of any one of paragraphs 1-4 wherein the cell suspension comprises granulocyte-macrophage colony-forming cell (GM-CFC).

6. The method of any one of paragraphs 1-5 wherein the cell suspension comprises megakaryocyte colony-forming cell (Mk-CFC).

7. The method of any one of paragraphs 1-6 wherein the cell suspension comprises T cell colony-forming cell (T-CFC) and/or B cell colony- forming cell (B-CFC).

8. The method of any one of paragraphs 1-7 wherein the cell suspension comprises colony-forming cell-blast (CFC-blast), high proliferative potential colony forming cell (HPP-CFC), macrophage colony-forming cell (M-CFC), granulocyte colony forming cell (G-CFC), colony- forming unit-erythroid (CFU-E), colony-forming cell-basophil (CFC-Bas) and/or colony-forming cell-eosinophil (CFC-Eo).

9. The method of any one of paragraphs 1-8 wherein each of the one or more samples is dispensed into three, four, five or six vessels. 10. The method of any one of paragraphs 1-9 wherein each of the one or more samples is dispensed in a fixed volume.

11. The method of paragraph 10 wherein the fixed volume is 100 μl.

12. The method of any one of paragraphs 1-10 wherein one or more growth factors are added to the one or more samples. 13. The method of paragraph 12 wherein the cell suspension comprises B-CFC and the growth factor comprises IL-7.

14. The method of any one of paragraphs 12-13 wherein the method further comprises adding a background control that does not contain the one or more growth factors.

15. The method of any one of paragraphs 1-14 wherein the cell suspension is derived from normal blood, peripheral blood, bone marrow or umbilical cord blood.

16. The method of any one of paragraphs 1-15 wherein the one or more vessels are wells in a 4 well Petri dish or in a 96 well microplate.

17. The method of paragraph 16 wherein the microplate is a luminescence plate.

18. The method of paragraph 17 wherein the luminescence plate is non-sterilized and non-coated

19. The method of any one of paragraphs 1-18 wherein the one or more cell suspensions is thawed from one or more frozen cell suspensions.

20. The method of any one of paragraphs 1-19 wherein the one or more cell suspensions comprises one or more stem cell subsets. 21. The method of paragraph 20 wherein the one or more stem cell subsets is selected from the group consisting of CD34, CD 117, CD 133 and CD90.

22. The method of any one of paragraphs 1-22 wherein the one or more cell suspensions has a concentration of about 0.5 x 10 6 cells/ml or about 0.5 x 10 4 cells/100 μl culture.

23. The method of any one of paragraphs 1-22 wherein the one or more cell suspensions is purified to CD34 + cells and has a concentration of about 1 x 10 4 cells/ml to about 2 x 10 4 cells/ml or about 1 x 10 2 cells/100 μl culture to about 2 x 10 2 cells/100 μl culture. 24. The method of any one of paragraphs 1-23 wherein incubating for 7 days at 37

C in a humidified atmosphere with 5% CO 2 and 5% O 2 , the one or more samples are transferred to a 23 C humidified incubator with 5% CO 2 and left to equilibrate at least 30 minutes.

25. The method of any one of paragraphs 1-24 wherein a minimum of 20 samples from each tissue type is analyzed.

26. The method of any one of paragraphs 1-25 wherein the serum mix comprises bovine serum albumin, an insulin, an iron-saturated transferrin, a serum and IMDM.

27. The method of any one of paragraphs 1-26 wherein the growth factor mix comprises at least one growth factor selected from the group consisting of erythropoietin, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L, and leukemia inhibitory factor, and combinations thereof.

28. The method of any one of paragraphs 1-27 wherein the methyl cellulose mix has between about 1.5% and about 2.5% methyl cellulose.

29. The method of any one of paragraphs 1-28 wherein the concentration of fetal bovine serum in the cell growth medium is between about 0% to about 10% by volume.

30. The method of any one of paragraphs 1-29 wherein the concentration of methyl cellulose in the cell growth medium is about 0.7% by weight. 31. The method of any one of paragraphs 1 -29 wherein the method further comprises contacting the target cell population with at least one cytokine.

32. The method of paragraph 31 wherein the at least one cytokine is selected from the group consisting of erythropoietin, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L, leukemia inhibitory factor, and combinations thereof.

33. The method of any one of paragraphs 1-32 wherein the one or more cell suspensions comprises hematopoietic stem cells and/or hematopoietic progenitor cells.

34. The method of paragraph 33 wherein the primary hematopoietic cells are isolated from an animal tissue selected from the group consisting of peripheral blood, bone marrow, umbilical cord blood, yolk sac, fetal liver, and spleen.

35. The method of paragraph 34 wherein the animal tissue is obtained from a human.

36. The method of paragraph 34 wherein the animal tissue is obtained from a mammal.

37. The method of paragraph 36 wherein the mammal is selected from the group consisting of cow, sheep, pig, horse, goat, dog, cat, non-human primates, rodents, rabbit and hare.

38. The method of any one of paragraphs 1-37 wherein the reagent capable of generating luminescence in the presence of ATP comprises luciferin and luciferase.

39. A kit for performing any one of the methods of paragraphs 1 to 38 comprising the compositions of any one of paragraphs 1 to 38 and instructions for performing the method of any one of paragraphs 1 to 38.

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Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.