Predictive Hematotoxicity /

Bone Marrow Toxicity

In Vitro High-Throughput Stem and Progenitor Cell Hematotoxicity Screening and/or Testing using HALO®-Tox HT

 

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The infographic above provides an overview of HemoGenix® hematotoxicity / bone marrow toxicity capabilities. 

For more information, please contact HemoGenix® directly at contractresearch@hemogenix.com or call (719) 264-6250. See our 100% ratings on the ThermoFisher Scientific Service Marketplace 

 

Click on the arrows to expand the accordions below

 

 

 

 

Any drug, compound agent or perturbation, (drug, environmental agent or xenobiotic, food addition, cosmetic addition, fuel addition, radiation or other exposure perturbation), has the potential to affect the normal steady state functioning and regulation of the blood-forming or hematopoietic system.

 

The hematopoietic system, like most biological systems in the body, is a stem cell system. The hematopoietic system is unique, however, in that it has the potential to produce multiple cell types, each with different functions, from a single stem cell. For all biological systems, the stem cell, or rather the stem cell compartment, which contains many different stages of stem cell primitiveness or maturity, is the foundation upon which all biological systems are built. Damage to the stem cell compartment, or any part of it, can mean damage to the biological system and the organism as a whole. 

 

Stem cell hematotoxicity refers to damage to the stem cell compartment that affects the rest of the lympho-hematopoietic system, its organization, regulation and feedback mechanisms that are required to maintain a steady state condition. 

 

Bone marrow toxicity, which is often referred to as myelotoxicity, means toxicological damage to one or more parts of the lympho-hematopoietic systems and microenvironmental system that supports it. 

 

From the viewpoint of toxicology, hematotoxicity and immunotoxicity are inseparable, since common stem cells give rise to both systems and, if these are damaged, both systems will be affected. It is for this reason why detecting the response of stem cells to potentially toxic agents can provide predictive information for the rest of the lympho-hematopoietic system. 

 

Conventional pathology to detect toxicity to the bone marrow and other hematopoietic tissues during pre-clinical animal testing does not provide the predictive insight of advanced in vitro toxicity testing platforms. 

During the late 1990s and early 2000s, the European Center for the Validation of Alternative Methods (ECVAM) published several articles on using the colony-forming cell (CFC) assay to predict drug-induced myelosuppression. The studies were initially focused on the myelomonocytic (granulocyte-macrophage) lineage and later the megakaryocytic lineage. These studies, performed at multiple locations, tried to validate the CFC assay in the hope of using it for bone marrow toxicity. Indeed, HemoGenix® was one of the first companies that provided (and still provides) this assay to study bone marrow toxicity. 

 

However, since the first publication of the CFC assay in 1966, the methodology has not changed significantly. Despite the use of recombinant growth factors and cytokines and the introduction of automated colony counting, the assay has remained the same with the same problematic errors associated with it. This is primarily due to the use of viscous methylcellulose to immobilize the cells and the subjective or algorithm-based counting of colonies using a microscope, both of which are far from perfect. Using methylcellulose is a highly, error-prone methodology since it cannot be dispensed accurately. As a result, typical CFC results are associated with high coefficients of variation (CV) that do not aid statistical relevance and the accurate estimation of inhibitory concentration (IC) values. 

 

In addition, although the production of colonies of cells requires that the cells producing the colonies must proliferate, the CFC assay does not quantitatively measure cell proliferation, but instead determines the ability of the cells to differentiate. Although proliferation and differentiation overlap, they are two completely separate biological processes and cannot be determined using the same assay readout. The CFC assay is whole dependent upon the cells in the colony being able to differentiate and mature so that the colonies can be identified as belonging to one or other hematopoietic lineage. In this way, the possibility of an agent causing neutropenia can be differentiated from that causing anemia or thrombocytopenia. 

 

Stem cells, on the other hand, exist as very rare populations (usually less than 0.1% of the total bone marrow cellularity). Due, in part, to the low plating efficiency of the CFC assay, it is not only unreliable and non-repeatable, but is also extremely insensitive to detecting rare cell populations, such as stem cells. With the exception of HemoGenix®, other companies offering the CFC assay do not even incorporate the full spectrum of growth factors that enable very primitive hematopoietic stem cells (CFC-GEMM, see below) to be detected. This means that the CFC assay cannot be reliably used to determine stem cell hematotoxicity. 

 

Despite all the problems associated with the CFC assay, the FDA, in its infinite wisdom, still recommends the use of this assay to detect potential myelosuppressive effects of an agent. In other words, their recommendations are based on data that is nearly 20 years old and does not take into account the response of the stem cells or modern technology. 

 

In 2002, HemoGenix® launched, with the help of a grant from the National Cancer Institute, the platform that is now considered the most advanced stem cell hematotoxicity and bone marrow toxicity platform available. Known as HALO®-Tox HT, the platform incorporates Suspension Expansion Culture™ (SEC™) Technology that makes using methylcellulose and the CFC assay, obsolete. The added ability to use high-throughput, liquid handler instrumentation provides the accuracy, sensitivity and precision necessary to detect and measure rare stem cell populations. The incorporation of ATP bioluminescence technology as the most advance and sensitive, non-radioactive, signal detection system available, also provides the standardization and FDA bioanalytical method validation necessary to determine reliable and repeatable, potential toxicity required by the biopharmaceutical industry. This, in turn, has allowed the establishment of HALO®-Tox HT measurement assurance so that the results obtained for this, or any other HemoGenix® ATP bioluminescence-based toxicity testing platform, to be trusted. With a high (>80%) in vitro to in vivo concordance, HALO®-Tox HT has become the most trusted hematotoxicity screening and testing platform available from early drug development to pre-clinical animal studies and beyond.
 

The majority of studies performed by HemoGenix® incorporate a Complete Service, Full Report that can be used for an IND application. More recently, Sponsors have requested a more streamlined study and studies involving high-throughput screening. For this reason, HemoGenix® now provides 3 types of study format. 

 

  1. Complete Service, Full Report Study: Fully customized study that includes the Study Plan, Draft Text and Final Text Report with QA audit.
  2. Rapid Toxicity Study: A customized study that includes the Study Plan and full protocol, raw results and graphical data in a single Excel Workbook. No formal text report and no QA audit is performed. This type of study is designed for early drug development. No interpretation or conclusions are provided.
  3. High-Throughput Screening Module (presently only available for human studies): A screening study is designed for high-throughput screening of compounds during ADME/Tox screening in multiples of 5 on specific cell populations to provide the most important ranking information. The full protocol, raw results and graphical data are provided in an Excel Workbook. No interpretation or conclusions are provided. This type of Screening Study is part of the ComparaTOX™ HT Platform.

All instrument-based assay platforms and methylcellulose CFC assays are available for use with the following species:

  • Human
  • Non-human primate (Macaca fascicularis or cynomologus (Cyno) and Macaca mulatta (Rhesus)
  • Dog
  • Rat (various strains)
  • Mouse (various strains)

Other species available and which might be of interest include:

  • Minipig
  • Pig
  • Sheep
  • Horse 
 
Tissues and Purified Cell Populations Available for Testing
  • Bone marrow (all species)
  • Peripheral blood (human and large animal species; please inquire about rodents)
  • Umbilical cord blood (human)
  • Purified stem cell populations (e.g. human CD34 , CD133 cells)
  • Purified human B lymphopoietic cells 
  • Other hematopoietic organs from animals (e.g. spleen, fetal liver, yolk sac)

 

PLEASE NOTE: We often are asked whether we can perform assays, especially methylcellulose CFC assays, using "whole" or red blood cell or plasma-reduced  bone marrow, cord blood or peripheral blood, since this is provided by other companies. We DO NOT suggest or recommend using this type of tissue preparation for any toxicity assays, let alone quality or potency assays. Using these preparations may result in a less costly study, but the assays will lack sensitivity, accuracy, precision, reliability and reproducibility causing a serious underestimation of the results leading to potential misinterpretation and conclusion of the data. It is for this reason why we highly recommend a minimum mononuclear cell (MNC) fractionation for most species so as not to loose important cellular information.  

Please Note that all 3 study types, (1) Complete Service, Full Report, (2) Rapid Toxicity Study and, (3) Screening Study are available using HALO®-Tox HT.

 

HALO®-Tox HT is a proprietary, fully standardized, high-throughput toxicity assay platform that has been validated according to FDA Bioanalytical Method Validation Guidelines. It incorporates two technologies:

  1. Suspension Expansion Culture™ (SEC™) Technology that allows (a) standardization and validation, (b) high sensitivity and accuracy, (c) high reliability and reproducibility, (d) rapid assay completion (5-7 days) and, (e) high-throughput capability. Indeed, HemoGenix® is the only company worldwide that provides high-throughput hematotoxicity testing. In contrast to the traditional colony-forming assay (CFC), HALO®-Tox HT does not employ methylcellulose and therefore obviates many of the drawbacks of the CFC assay (see below).
  2. ATP Bioluminescence Technology. Measuring intracellular ATP using a luciferin/luciferase bioluminescence output has been employed by the biopharmaceutical industry for many years. HemoGenix® has taken this technology to a higher level by providing standardization, validation and measurement assurance parameters for all contract research studies. 

 

The nomeclature used to describe different cell populations of the lympho-hematopoietic systems is often confusing. In addition, although HALO® incorporates similar growth factor cocktails to detect different populations, colonies of cells are not produced, because HALO®-Tox HT incorporates advanced Suspension Expansion Culture™ (SEC™) Technology that obviates the use of methylcellulose and allows for high-throughput screening. Since the cell populations are detected in suspension culture, a different nomenclature is used to describe the populations:  "SC" means "stem cell" and "P" means "progenitor cell" for all populations detected using HALO®-Tox HT.

 

 

Stem Cell Populations Detected using HALO®-Tox HT
Stem Cell Type

HALO®

Stem Cell($)

Designation

Growth Factor Cocktail

Pathways

Predictive

Toxicity

HALO®-Tox HT Screening or Testing

Quiescent, primitive lympho-hematopoietic stem cell: High Proliferative Potential - Stem and Progenitor Cell

SC-HPP 1

IL-3, IL-6, SCF, TPO, Flt3-L Lympho-Hematopoietic Yes Testing only
Induced, primitive lympho-hematopoietic stem cell: High Proliferative Potential - Stem and Progenitor Cell

SC-HPP 2

EPO, GM-CFC, IL-2, IL-3, IL-6, IL-7, SCF, TPO, Flt3-L Lympho-Hematopoietic

Anemia

Neutropenia

Thrombo-cytopenia

Lymphopenia

384-well HT screening(*) and normal testing
Primitive hematopoietic stem cell: Granulocyte, Erythroid, Macrophage, Megakaryocyte

SC-GEMM 1

EPO, GM-CFC, IL-3, IL-6, SCF, TPO, Flt3-L Hematopoietic only

Anemia

Neutropenia

Thrombo-cytopenia

384-well HT screening(*) and normal testing

 (*) Part of the HemoGenix® ComparaTOX™ HT Platform for the rapid screening of compounds in multiples of 5. Please contact HemoGenix® for more nformation.

($) Other stem cell populations are also available. Please contact HemoGenix™ for more information.

 

 

 

Progenitor Cell Populations Detected Using HALO®-Tox HT

Progenitor Cell Type

HALO®

Progenitor Cell Designation

Growth Factor Cocktail Pathways Detected Predictive Toxicity

HALO®-Tox HT Screening or Testing

Primitive erythropoietic

progenitor cell: Stem cells

induced into the erythropoietic

lineage

P-BFU 1

EPO, IL-3, SCF Erythropoietic lineage Anemia 384-well HT screening(*) and normal testing

Primitive erythropoietic

progenitor cell: Primitive

erythropoietic progenitors

already in compartment

P-BFU 2

EPO alone Erythropoietic lineage Anemia Testing only

Primitive granulocyte-

macrophage (GM) progenitor:

Stem cells induced into the

GM lineage

P-GM 1

GM-CSF, IL-3, SCF

Granulocyte

/macrophage lineage

Neutropenia 384-well HT screening(*) and normal testing

Primitive granulocyte-

macrophage progenitor:

Primitive GM progenitors already

in compartment

P-GM 2

GM-CSF, G-CSF, IL-3, SCF

Granulocyte

/macrophage lineage

Neutropenia Testing only

Primitive megakaryocyte

progenitor: Stem cells induced

into the megakaryopoietic

lineage

P-Mk 1

TPO, IL-3, SCF

Megakaryo-poietic lineage Thrombo-cytopenia 384-well HT screening(*) and normal testing

Primitive T-lymphocyte

progenitor: Stem cells induced

into the T-cell lineage

P-Tcell 3

IL-2 or cocktail

T-cell lineage Lymphopenia 384-well HT screening(*) and normal testing

Primitive B-lymphocyte progenitor: Stem cells induced

into the B-cell lineage

P-Bcell 2

IL-4 + cocktail

B-cell lineage Lymphopenia 384-well HT screening(*) and normal testing

 (*) Part of the HemoGenix® ComparaTOX™ HT Platform for the rapid screening of compounds in multiples of 5. Please contact HemoGenix® for more nformation.

 

In addition to screening or testing individual cell populations using HALO®-Tox HT, HemoGenix® has developed a "Global" Toxicity Assay Platform for testing compounds on multiple cell populations simultaneously to provide a more detailed "global" assessment of the lympho-hematopoietic lineages affected by potential toxicity. 

 

The HALO®-Tox HT "Global" Platform is available either as (1) A Complete Service, Full Report, or (2) A Rapid Toxicity Study.

 

HALO®-Tox HT "Global" Contract Services are available for the following species:

  • Human
  • Primate (Cyno and Rhesus)
  • Dog
  • Rat
  • Mouse 

 

There are three (3) options for the HALO®-Tox HT "Global" Platform. These are:

1. HALO®-Tox HT "Global" 4-Population Assay that mesures the response to:

  • SC-GEMM 1
  • P-BFU 1
  • P-GM 1
  • P-Mk 1

 

2. HALO®-Tox HT "Global" 5-Population Assay that measures the response to:

  • SC-HPP 1
  • SC-GEMM 1
  • P-BFU 1
  • P-GM 1
  • P-Mk 1 

 

3. HALO®-Tox HT "Global" 7-Population Assay that measures the response to:

  • SC-HPP 1
  • SC-GEMM 1
  • P-BFU 1
  • P-GM 1
  • P-Mk 1
  • P-Tcell 3
  • P-Bcell 2* 
(*) This population requires that a purified population of P-Bcell are used. 

Validation of HALO®-Tox HT as a Cytotoxicity Assay 

Besides multiple biopharmaceutical companies validating HALO® either in-house or by contract services, HALO® has been validated against the Registry of Cytotoxicity Prediction Model. The Registry of Cytotoxicity contains a list of compounds for which the LD50 values in rats or mice have been documented. If the LD50 for a group of reference compounds is plotted against the IC50 values obtained from an in vitro assay, in this case HALO®-Tox HT, the individual points will lie on a linear regression with specific parameters. This validates the assay as a cytotoxicity assay. Thus, HALO®-Tox HT has been validated against the Registry of Cytotoxicity.

 

Validation of HALO-Tox HT using the Registry of Cytotoxicity Prediction Model 

However, there is an even more interesting aspect. This is shown in the table below. Using the Registry of Cytotoxicity Prediction Model, it is possible to convert in vitro IC50 values into clinically relevant doses, either in mg/kg, mg/m2 or as mg/day. The conversion of in vitro IC values obtained using HALO®-Tox HT have been converted into clinically relevent doses based on the Registry of Cytotoxicity Prediction Model. In the majority of cases, the predicted in vitro values correspond to the dose ranges used in the clinic to treat various forms of human cancer. That is, the IC50 values derived from HALO®-Tox HT are, in the majority of cases, in the same order of magnitude as those used for treatment. It should be noted that this model can also be used to to predict the starting doses for pre-clinical animal studies. 

 

Estimation of Clinical Starting Doses Using HALO-Tox HT 

HALO®-Tox HT has been validated according to FDA Bioanalytical Method Validation Guidelines which are summarized below. 

 

Validation Parameters for HALO-Tox HT

 

Validation of the HALO®-Tox HT Platform was only possible by incorporating controls to calibrate the assay and standards to ensure that the assay works correctly prior to processing and determining samples.  

After assay validation, it was then possible to establish measurement assurance parameters from historical data that ensure results are trustworthy.  

Measurement Assurance Parameters for HALO-Tox HT

 

Example 1. HALO®-Tox HT and Multiple Donors - A Case for Reproducibility

The diagram below shows the response of primitive human hematopoietic stem cells (SC-GEMM 1) to cycloheximide by 3 separate human donors performed on different days. Although each donor exhbits a different number of responding cells, as indicated by the different starting ATP concentrations at the lowest cycloheximide dose, the shape of the dose response curve and the IC50 values are very similar between each donor. This example demonstrates the high degree of reproducibility using HALO®-Tox HT. The example also illustrates that, in the majority of cases, a single donor is sufficient to obtain reliable results, thereby significantly reducing the cost of a study. 

 

Response of 3 human bone marrow donors to cycloheximide using HALO-384 HT   

Example 2. Species Comparison

It is often the case that one compound will exhibit a different response in different species. The graphs below demonstrate how this may, or may not, occur. Although HALO®-Tox HT can predict many aspects of potential hematotoxicity, it cannot, as yet, predict differences in response between species.  

 

Comparison of species toxicity response using HALO-Tox HT

 

Example 3. High-Throughput Screening Using HALO®-384 Tox HT

The following shows a study in which up to 12 compounds, with different targets, were tested on human hematopoietic stem cells (SC-GEMM1 ) and granulocyte-macrophage progenitor cells (P-GM 1) using HALO®-384 Tox HT high-throughput screening platform. All of the dose response curves are fitted to a 4-parameter logistic curve fit using SoftMax Pro software. The approximate IC50 values are prvided by Parameter C. 

The compounds shown for the P-GM 1 response were the same as those used by Pessina and coworkers at ECVAM to obtain the maximum tolerated dose (MTD) at IC90, in order to try and validate the colony-forming unit (CFU, see below) assay for toxicity testing. Although results were similar between the assays, HALO®-384 Tox HT is not only more rapid, accurate and reliable, but can also be used to rapidly rank compounds based on compound type, cell type and species. HALO®-Tox HT is part of the HemoGenix® ComparaTOX™ Platform.   

 

High-Throughput Screening Using HALO-384 HT

 

Example 4. Predicting Hematotoxicity Using HALO®-Tox HT "Global"

The HALO®-Tox HT "Global" Platform provides all the information to predict hematotoxicity for different compound types and species. As described above, there are 3 HALO®-Tox HT "Global" Platforms:

  • HALO®-Tox HT "Global" 4-Population
  • HALO®-Tox HT "Global" 5-Population 
  • HALO®-Tox HT "Global" 7-Population

The example below shows an example of the HALO®-Tox HT "Gloabl" 7-Population Platform for Mitomycin-C using huamn bone marrow mononuclear cells (MNC) as the target cell population. Notice that there are 3 groups of response, namely (1) the stem cell response, (2) the hematopoietic progenitor cell response and, (3) the lymphopoietic response.  

 

HALO-Tox HT "Global" 7-Population Hematotoxicity Platform

 

Example 5. In Vitro to In Vivo Concordance

The ability to predict the in vivo response from in vitro data has been one of the foundations for using in vitro assays to measure and determine potential toxicity of drugs and xenobiotics. In 2009, Olaharski and his colleagues published data showing that HALO®-Tox HT provided an 82% concordance with in vivo data from the rat. However, it should be noted that based on decades of publications, an extremely high concordance between in vitro and in vivo data for for multiple hematopoietic applications has been shown.  

In Vitro to In Vivo Concordance Using HALO-Tox HT  

HemaToxFlow™ is an in vitro flow cytometric assay used to determine potential hematotoxicity of stem and progenitor cells from human and mouse tissues. Since HemaToxFlow™ uses CD markers to define specific differentiated cell populations, it can replace the use of colony-forming cell (CFC) assays which relies on the ability of primitive cells to differentiate and mature.  

 

For human studies, HemaToxFlow™ utilizes mononuclear cells (MNC), CD34+ or CD133+ cells from:

  • Bone marrow
  • Umbilical cord blood
  • Peripheral blood (normal or mobilized).  

 

For murine studies, fractionated and purified cells can be used, but are not a requirement. Murine target cells:

  • Bone marrow
  • Spleen
  • Fetal liver.  

 

HemaToxFlow™ reagents are used to stimulate the following defined cell populations from both species:

  • SC-GEMM 1: Primitive hematopoietic stem cell
  • P-BFU 1: Primitive erythropoietic progenitor cell
  • P-GM 1: Primitive granulocyte-macrophage progenitor cell
  • P-Mk 1: Primitive megakaryopoietic progenitor cell. 

 

Method Summary

HemaToxFlow™ is performed in 96-well plates.

  1. Cell suspensions are prepared and adjusted to the correct final concentration.
  2. Test compounds are dissolved and serial dilutions prepared.
  3. HemaToxFlow™ Reagent for the specific cell population or populations to be analyzed are dispensed into the 96-well plate(s) using a liquid handler.
  4. This is followed by the addition of the cell suspension to each well.
  5. The test compound or compounds represent the final addition, also performed by a liquid handler.
  6. The plates are incubated in a 37 oC incubator for 4-5 days for murine cells and 5-7 days for human cells.
  7. After incubation, the cells in the plates are centrifuged and the supernatant removed and discarded.
  8. Pre-diluted, specific fluorophore-conjugated antibodies are then added in panels to each well so that different cell populations can be detected. Counting bead are also added to determine cell number.
  9. The cells in each well of the plate are then analyzed by flow cytometry using a Beckman Coulter Cytoflex flow cytometer.
  10. Cell counts are ploted against the drug dose and a 4-parameter logistic curve fit is used to determine IC values.
  11. If required, HemaToxFlow™ can be verified against HALO™-Tox HT

 

The table below shows the CD antibodies used to detect different cell populations from different human and mouse using the HemaToxFlow™ assay. 

 

HemaToxFlow™ Assay Antibodies Cell Population Detected
Human Stem Cells* CD34
CD133
 

Hematopoietic stem cells
Primitive hematopoietic stem cells

(Additional antibodies can be added to determine lineage-specific differentiated cells).

Human Erythroid Cells CD235a (GlyA) Erythroid cells
Human Myelod Cells  CD13
CD14
CD15
Myelod progenitor cells
Monocytes
Granulocytes
Human Megakaryocytes CD41
CD61
Megakaryocytes
Platelets
     
Murine Stem Cells*  CD34 Hematopoietic stem cells
Murine Erythroid Cells CD235a
Ter-119
Erythroid cells
Murine Myeloid Cells Mac-1/CD11b
F4/80
CD66b
Gr-1/Ly6G
Ly6C
Monocytes/macrophages
Monocytes/macrophages
Granulocytes
Granulocytes
Granulocytes
Murine Megakaryocytic CD41
CD61
Megakaryocytes/platelets
Megakaryocytes/platelets

(*) Due to the fact that the SC-GEMM 1 population is stimulated with a growth factor cocktail that produces lineage-specific hematopoietic cells, the CD antigen markers used for progenitor cell differentiation can also be used to determine differentiated cells produced from the stem cell population.

In contrast to HALO®-Tox HT, which quantitatively measures functional viability and cell proliferation, or the lack of it due to cytotoxicity, the methylcellulose colony-forming cell (CFC) assay relies on the differentiation and maturation of cells, grown in colonies, in order to identify the cell that gave rise to the colony. Although the CFC assay has been used for many decades, it is not a robust, reliable or repeatable assay. Nevertheless, it is often requested and provided for toxicity studies. 

 

HemoGenix® has developed three formats for the CFC assay all of which are available for contract services.

  • ColonyGro™: A traditional 35mm Petri dish format performed using a minimum of duplicate replicates and a total culture volume of 1mL/dish. Colony enumeration is manual and occurs after 12-14 days incubation.
  • CAMEO™-4: A miniaturized version of ColonyGro™ performed in quadruplicate using 4 wells incorporated into a 35mm Petri dish. The total assay volume is 0.1mL/well and the incubation time is between 9 and 12 days. Colony enumeration is manual.
  • CAMEO™-96: Originally the first 96-well methylcellulose hematotoxicity assay developed in 2002, CAMEO™-96 is both a proliferation and differentiation assay. Cultures for CFC are setup in 96-well plates and colonies are counted between 4 to 7 days. The proliferation within the colonies is then quantitatively measured using the same standardized and validated ATP bioluminescence readout used for HALO®-Tox HT. The number of colonies correlates directly with the intracellular concentration of ATP. This correlation allows the CFC part of the assay to be standardized. Thus, CAMEO™-96 is the only true standardized CFC assay available.

 

All CFU assays are available either as (1) A Complete Service, Full Report, or (2) A Rapid Toxicity Study. 

 

Cell Type

CFC Population

Growth Factor Cocktail

Pathways

Predictive

Toxicity

CFC Assays Available

Primitive hematopoietic stem cell: Granulocyte, Erythroid, Macrophage, Megakaryocyte

CFC-GEMM 1

EPO, GM-CFC, IL-3, IL-6, SCF, TPO, Flt3-L Hematopoietic only

Anemia

Neutropenia

Thrombo-cytopenia

ColonyGro™

CAMEO™-4

CAMEO™-96

Primitive erythropoietic progenitor cell: Stem cells induced into the erythropoietic lineage

BFU-E 1

EPO, IL-3, SCF Erythropoietic lineage Anemia ColonyGro™
CAMEO™-4
CAMEO™-96
Primitive erythropoietic progenitor cell: Primitive erythropoietic progenitors already in compartment

BFU-E 2

EPO alone Erythropoietic lineage Anemia ColonyGro™
CAMEO™-4
CAMEO™-96
Primitive granulocyte-macrophage (GM) progenitor: Stem cells induced into the GM lineage

GM-CFC 1

GM-CSF, IL-3, SCF (similar to H4534)

Granulocyte

/macrophage lineage

Neutropenia ColonyGro™
CAMEO™-4
CAMEO™-96
Primitive granulocyte-macrophage progenitor: Primitive GM progenitors already in compartment

GM-CFC 2

GM-CSF, G-CSF, IL-3, SCF

Granulocyte

/macrophage lineage

Neutropenia ColonyGro™
CAMEO™-4
CAMEO™-96
Primitive megakaryocyte progenitor: Stem cells induced into the megakaryopoietic lineage

Mk-CFC 1

TPO, IL-3, SCF

Megakaryopoietic lineage Thrombocytopenia ColonyGro™
CAMEO™-4
CAMEO™-96
Erythropoietic precursor cell: Colony-Forming Unit - Erythroid CFU-E EPO alone Erythroid lineage Anemia ColonyGro™
CAMEO™-4
Granulocyte precursor cell: Granulocyte - Colony-Forming Cell G-CFC G-CSF Granulocyte lineage Neutropenia ColonyGro™
CAMEO™-4

Macrophage precursor cell: Macrophage - Colony-Forming Cell

M-CFC M-CSF Macrophage lineage Monocytopenia ColonyGro™
CAMEO™-4

 

Since 1966, the colony-forming unit (CFU) or colony-forming cell (CFC) assay has, with few exceptions, remain unchanged. These exceptions include the change from agar to water-soluble methylcellulose and the incorporate of recombinant growth factors and cytokines. Otherwise, the format of the CFU assay has removed the same for over half a century. There has been little incentive to improve the assay. As a result, the CFU assay has never been validated because no controls or standards exist for the assay. As far as toxicity testing is concerned, this lack of validation means that the results from the assay, regardless of the application for which it is used, are untrustworthy. It was for this reason that HALO®-Tox HT was developed with the help of an SBIR Phase I and Phase II grant from the National Cancer Institute (NCI).  

 

Although HemoGenix® provides the CFU assay as a contract research service (see above), the majority of biopharmaceutical companies now rely on the HALO®-Tox HT Platform for assessing potential hematotoxicity. The following diagrams show a comparison between HALO®-Tox HT and the CFU assay for the response of hematopoietic progenitor cells to cycloheximide for human, non-human primate, rat and mouse bone marrow.  

 

Comparison of CFU and HALO-384 HT Cycloheximide Response for Human Bone Marrow 

 Comparison of CFU and HALO-384 HT Cycloheximide Response for Rat Bone MarrowComparison of CFU and HALO-384 HT Cycloheximide Response for Rat Bone Marrow

Comparison of CFU and HALO-384 HT Cycloheximide Response for Mouse Bone Marrow 

 

Besides the basic assays used to determine stem cell hematotoxicity and general bone marrow toxicity, HemoGenix® also offers a number of other assays that can be multiplexed with HALO® and CAMEO™ assays or used individually. These can be incorporated to study mechanism of action. For more information, please refer to the Mechanism of Action Page.

Analyzing Mechanism of Action of
Bone Marrow Toxicity and Stem Cell Hematototoxicity 

Assay Name Assay Type Pathway Readout

HALO®-96 PRT

(see below)

Residual stem cell toxicity and change in compound sensitivity Proliferation Bioluminescence

HALO® Real Time

Kinetics of compound response Proliferation Bioluminescence
Cell cycle DNA marker Proliferation Fluorescence
FlowDiff™ Hematopoietic membrane expression markers Characterization / Differentiation Fluorescence
LIVEGlo™ iATP Metabolic viability / Mitochondrial Function Bioluminescence
Dye Exclusion Viability 7-Aminoactinomycin D (7-AAD) / Propidium Iodide (PI) Membrane integrity Fluorescence
Mitochondrial ToxGlo™* ATP Mitochondrial dysfunction Bioluminescence / Fluorescence
Glutathione Assay Glutathione Oxidative stress Luminescence

OxyFLOW™

8-Oxoguanine addudcts Oxidative DNA damage Fluorescence
CaspaseGlo™* Apoptosis Caspases Luminescence
Annexin-V / PI Apoptosis / Necrosis Phosphatidylcholine Fluorescence
GFkine™  Growth factor, cytokine production / release Multiple Multiple

* Promega Corporation assays 

Residual stem cell toxicity occurs when a drug or other agent leaves some stem cell unharmed or undamaged. If the insult is only administerd once, there is a chance that the system may recover. However, multiple doses may not only damage stam cells that were not initially affected, but may also induce increased sensitivity to the drug on multiple dosing.

HALO® PRT: Measuring Residual Toxicity

There are several permutations, but the basic methodology occurs as follows:

  1. Perform a drug or compound dose response on one or more stem cell populations within the stem cell compartment in a primary in vitro cell culture system.
  2. Analyze the results to determine if residual stem cells remain after the first treatment.
  3. Remove cells treated with different doses from the primary cell culture and perform a secondary, re-plating culture of the treated cells.
  4. Analyze the secondary response for changes in dose sensitivity and expansion potential.

How HALO-96 PRT can be used to measure residual hemotoxicity

Click image for larger view


Assays to Determine Predictive Residual Toxicity (PRT)

HALO® PRT is used to predict residual toxicity. An example of a PRT assay is shown. In this example, residual toxicity is determined for Daunorubicin. In the first, primary culture, a Daunorubicin dose response is performed for both the primitive lympho-hematopoietic (SC-HPP) and more mature hematopoietic (SC-GEMM) stem cells. After secondary re-plating of the cells from the HPP-SP dose response, the results shown in the right-hand graph were obtained. In this case, both the SC-HPP 1 and SC-HPP 2 primitive stem cell populations were detected. The SC-HPP 2 assay detects the ability of the HPP-SP 1 cells to expand. Since Daunorubicin results in eradication of the both primitive and mature stem cell populations at high doses (left graph), it is clear that few if any residual cells will remain as depicted in the right-hand graph. However, it is also clear that the SC-HPP stem cell population has increased its sensitivity to the drug at all IC values. 

The interaction of drugs on the cytochrome P450 system (activation and inhibition) in hepatocytes is the traditional method of analyzing drug-drug interactions. However, these enzymatic reactions often do not correlate with the cellular effects that occur in a tissue. The HALO®-Tox DDI Platform is a one-of-a-kind assay to study the effects of drug-drug interactions. These are relatively complicated studies to perform since two or more compounds have to be titrated against each other.

 

Related to drug-drug interactions are the effects of drugs used in combination with each other. Such combinations are used in chemotherapy to treat different forms of cancer. Combination drug therapies are often more effective than single drugs. New drugs designed to be less toxic on their own could interact with more conventional drugs to be more toxic and even less effective. Such combinations can be analyzed to help predict unwanted and unnecessary side effects.

 

Please navigate to the DDI Page for more information.

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