Phone: (719) 264-6250    email:    Sign In | User Pages

Assays You Can Trust. Innovative Expertise You Can Count On.

Contract Services

Predictive In Vitro Bone Marrow Toxicity

and Stem Cell Hematotoxicity


Bone Marrow Toxicity/Stem Cell Hematotoxicity performed at HemoGenix 

Bone Marrow Toxicity/Stem Cell Hematotoxicity performed at HemoGenix


 HALO®-Tox HT: The "Gold Standard" for In Vitro Predictve Hematotoxicity Testing


The drug development pipeline and how HALO-Tox HT can be used at different stages

Click image for larger view

HALO®-Tox HT forms the backbone of hematotoxicity / hemotoxicity contract services performed by HemoGenix®. Standardized and fully validated, HALO®-Tox HT is a high-throughput, ATP bioluminescence screening and multiplexing platform that has been shown to exhibit greater than 80% concordance between in vitro and in vivo data (Toxicol Let. 2009; 188/2:98-103). The concept and principle underlying the measurement of hematopoietic cell populations using ATP bioluminescence is described elsewhere.   
The Benefits of Using HALO®-Tox HT for Predictive In Vitro Hematotoxicity Testing
  • Fully validated in vitro assay platform used by small, medium and the largest biopharmaceutical companies and government agencies.
  • Highly predictive; greater than 80% concordance between in vitro and in vivo results.
  • High-throughput capability using 96- or 384-well plate formats allows ADME-Tox drug or compound screening, thereby significantly reducing unexpected results during pre-clinical testing.
  • Can be used at all stages of drug development, even during clinical trial patient monitoring.
  • 3Rs Alternative Assay Platform-Reduction, Refinement, Replacement for animal testing.
  • Available for multiple species comparisons.
  • Incorporates the most sensitive ATP bioluminescence readout available to measure proliferation, cytotoxicity, cell number and even apoptosis.
  • Uses proprietary Suspension Expansion Culture™ (SEC™) and Bioluminomics™ Technology.
  • Available for up to 7 different individual stem cell populations and 7 individual progenitor cell populations.
  • Also available for “global” testing to predict potential toxicity to the whole lympho-hematopoietic system. Measure 4, 5 or 7 cell populations simultaneously.
  • Results usually in 4 to 5 days depending on species.
  • Validated according to FDA Bioanalytical Method Guidelines and against the Registry of Cytotoxicity Prediction Model as a cytotoxicity assay and allows estimates of starting doses for animal and human studies from in vitro IC values.
  • Designed for multiplexing with other assay using the same sample.
  • Also available as assay kits that include everything required to perform the assay, except cells.
 HALO®-Tox Real Time: How long does it take for toxicity to be initiated?

Whereas HALO®-Tox HT is an end-point assay, HALO®-Tox RT is used to follow the onset of potential toxicity in a dose-penedent manner. HALO®-Tox RT allows the kinetics of cytotoxicity to be established and helps build a profile of the drug or compound to understand its efficacy and potential toxicity.


Like HALO®-Tox HT, HALO®-Tox RT is a bioluminescence assay, but does not use changes in intracellular ATP concentration to measure cell proliferation or inhibition. Instead, HALO®-Tox RT incorporates Promega's RealTime-Glo™ MT reagents that uses NanoLuc® luciferase and a proprietary prosubstrate allowing the cells to demonstrate their reducing potential and therefore their viability. HemoGenix® has incorported these reagents into its HALO®-Tox RT platform for real time contract services.


The non-destructive assay can be multiplexed with other readouts, especially flow cytometric phenotypic analysis to analyze the cell population(s) being affected.


Benefits of Using HALO®-Tox Real Time (RT) for Predictive In Vitro Hematotoxicity Testing and Studies on the Blood-Forming System
  • Allows potential cytotoxicity to hematopoietic stem and/or progenitor cells to be detected at the earliest possible timepoint.
  • HALO®-Tox RT can be used to analyze compounds that are not cytotoxic, but rather potentiate or stimulate hematopoietic cells.
  • HALO®-Tox RT is available in both 96- and 384-well high throughput formats.
  • Uses proprietary Suspension Expansion Culture™ (SEC™) Technology in low serum and serum-free formats to culture hematopoietic cells.
  • Can be used simultaneously with HALO®-Tox HT as a more sensitive end-point readout assay.
  • HALO®-Tox RT is a non-lytic assay that can be simultabneously multiplexed with flow cytometric phenotypic analysis to determine which cells are affected.
  • HALO™-Tox RT is available to detect up to 7 stem cell and 9 hematopoietic progenitor cell populations from 8 different species. 

 The Colony-Forming Cell (CFC) Assay

The CFC assay is a clonal cell culture that has been used in hematopoietic research since it was first reported in 1966. The European Center for the Validation of Alternative Methods (ECVAM) used the methylcellulose CFC assay to study myelotoxicity and myelosuppresive agents. Primarily a manual, colony-counting assay, it is used primarily as a differentiation assay, since the cells that comprise the colonies can be morphologically identified, thereby providing information of the cell type that produced the colony.


The CFU assay is usually used to determine the effect of agents on the granulocyte-macrophage, erythropoietic and/or megakarypoietic lineages to provide information on potential neutropenia, anemia and/or thrombocytopenia, respectively. 


The HALO® Platform was originally derived from the traditional, methylcellulose CFC assay performed in 35mm Petri dishes. HemoGenix now has 3 formats of the CFC assay:

  • ColonyGro™ is the equivalent of the traditional methylcellulose CFC assay that can be used to study the differentiation ability of up to 19 different cell populations from 8 different species. ColonyGro™ is usually performed in duplicate or triplicate in a total volume of 1mL. Colonies are usually enumerated after 12 days of incubation.
  • CAMEO™-4 is a miniaturized version of the ColonyGro™ assay. It is performed in quadruplicate in wells containing a volume of 0.1mL. Colonies are enumerated after 9-10 days of incubation. Development of CAMEO™-4 led to the development of the next interation of the CFC assay.
  • CAMEO™-96 is a methylcellulose CFC assay performed in 96-well plates. Re-developed from the ground up, CAMEO™-96 is used where both cell proliferation and differentiation response to compound administration have to be measured. Colonies are first counted in each well. This is followed by the addition of an ATP Enumeration Reagent and processing of the cultures in the same manner used for any of the HALO® assay. In fact, CAMEO™-96 was the forerunner to the HALO® Platform.


Like ColonyGro™, CAMEO™-4 and CAMEO™-96 can be used to study multiple cell populations from up to 8 different species (see below). In addition, all 3 assays can be multiplexed with other assay readouts, including flow cytometry.

 Species Available for Bone Marrow Toxicity / Hematopoiesis Studies

All three assay platforms 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)
Cells and Tissues Used for Hematotoxicity Testing
  • Bone marrow (all species)
  • Peripheral blood (human and large animal species)
  • Umbilical cord blood (human)
  • Purified stem cell populations (e.g. human CD34 , CD133 cells)
  • Other hematopoietic organs from animals (e.g. spleen, fetal liver, yolk sac)

 Lympho-Hematopoietic Cell Populations Available for Enumeration

To learn more about the blood-forming system and how it is organized, please click here. A total of 7 different stem cell population, 9 different progenitor cell populations and 3 different precursor cell populations can be enumerated with HemoGenix® assay platforms. To see all of the blood-forming cell populations that can be detected and measured, please click here

 The Hematotoxicity Assay Panel - Mechanism of Action (MOA)

Individual or multiple cell populations can be analyzed by multiplexing with one or more of the assays listed below. All of the hematopoietic assay platforms have the flexibility to multiplex with individual or any combination of assay readouts below from a single sample. To this end, HemoGenix® has put together a panel of tests and assays that can be include separately or together in order to study both potential cytotoxicity and mechanism of action in a single study. For more information, please refer to the Mechanism of Action Page.

Hematopoietic / Hematotoxicity Assay Panel 

Assay Name Assay Type Pathway Readout
HALO®-Tox HT Intracellular ATP (iATP) Proliferation Bioluminescence
HALO®-Tox Real Time (RT) Reduction potential Proliferation Bioluminescence


(see below)

Residual stem cell toxicity and change in compound sensitivity Proliferation Bioluminescence
Cell cycle DNA marker Proliferation Fluorescence
CAMEO™-96 Clonal (colony formation) & iATP  Proliferation and Differentiation Colony number / Bioluminescence
CAMEO™-4 Clonal (colony formation) Differentiation Colony number (manual)
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


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 

 Examples of Stem Cell Hemotoxicity



Examples of measuring drug toxicity to hematopoietic cells using HALO-Tox HT

Click image for larger view

The diagram to the left shows the dose response relationship between a number of familiar drugs and the effect on in vitro hematopoietic multipotential mature stem cells, CFC-GEMM, derived from human bone marrow mononuclear cells. These results were obtained using HALO®-384 HT. The upper graph shows that Omeprezole, Cyclosporine, Cimetidine and Warfarin have little if any effect on hematopoietic stem cells over the dose range studied. In contrast, Tamoxifin shows partial cytotoxicity at high doses, while Verapamil is cytotoxic at high doses. The lower graph demonstrates that while AZT and Cisplatin produce an inhibition at the highest doses, both 5-Fluorouracil and Mitomycin-C are highly toxic.



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

Click image for larger view


How Results from HALO®-Tox HT Help Estimate in vivo Starting Doses?

Click image for larger view

Can Results from HALO®-Tox HT Help Estimate in vivo Starting Doses?

The answer to this question is, yes. Not only is HALO® one of the most predictive in vitro toxicity assays, available, but can also be used to estimate drug dosing both in animals and humans. This is because 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®, the points should allow a linear regression to be drawn with specific parameters. This not only allows the assay to be validated as a cytotoxicity assay, but allows an even more interesting aspect to be investigated. This is shown in the table. Using the Registry of Cytotoxicity Prediction Model, it is possible to convert in vitro IC50 values into clinically relevant doses, either in mg/kg or as mg/m2. The results of this conversion are shown in the table on the left. The doses on the far right of the table, are those used to treat various forms of human cancer. It can be seen that the estimated doses for the anti-cancer drugs shown and obtained using the IC50 values derived from HALO® are in the same order of magnitude as those used for treatment. Only one drug, namely vinblastine, is the exception. 

 How the HALO®-Tox HT "Global" Predictive Hematotoxicity is Performed

How HALO-Tox HT can be used to predict "global" hemotoxicity

Click image for larger view

HemoGenix® has designed 3 "global" predictive hematotoxicity panels that provide information on the hematopoietic alone or lympho-hematopoietic systems combined. These 3 "global" panels are:

Panel 1. 4-Population "Global" Panel: CFC-GEMM 1, BFU-E 1, GM-CFC 1
and Mk-CFC 1.

Panel 2. 5-Population "Global" Panel: HPP-SP 1, CFC-GEMM 1, BFU-E 1,
GM-CFC 1 and Mk-CFC 1.

Panel 3. 7-Population "Global" Panal: HPP-SP 1, CFC-GEMM 1, BFU-E 1,
GM-CFC 1, Mk-CFC 1, T-CFC and B-CFC.
The response of the stem cells to a drug or agent provides predictive information on the effects observed downstream. The addition, the response of the early progenitor cells provides an added "global" view of the whole system indicating whether one lineage is affected more than another and ranking the cell types according to the compound response.

An example of "global" predictive hematotoxicity testing is shown for Daunorubicin. Although the 7-population panel is shown, the response of the quiescent primitive stem cell population (HPP-SP 1) is also demonstrated. The results from this population indicate that, even in a mostly quiescent state, very primitive stem cells can be affected by drugs and other compounds. Action of a compound at this very primitive stage of lympho-hematopoiesis will have an affect on the whole system and could be associated with drastic repercussions.

 How Residual Stem Cell Toxicity and Changes in Drug Sensitivity Studies can be Studied

A drug or other insult may affect stem cells in different ways. The effect may be felt on stem cells that are quiescent as well as those in cell cycle and proliferating. Small molecules can enter quiescent stem cells. When called upon to enter the cell cycle and proliferate, the effect of the agent may partially or completely inhibit this function. After treatment of quiescent or proliferating stem cell populations, it may be noticed that not all of the stem cells are affected. This may be due to resistance to the agent or perhaps a proportion of the stem cells were in a cell cycle phase in which they were unaffected by the treatment. The importance of this residual stem cell population lies in the fact that these stem cells could repopulate or replenish the system if allowed to do so. However, changes to the residual stem cells may have occurred. These could include potential changes in drug or compound sensitivity and/or the ability of the residual stem cells to expand after one or more treatments. These are important factors in understanding the response and toxicity to stem cells. For this reason, HemoGenix® developed Predictive Residual Toxicity Assays that are highly specialized assays for stem cell systems.

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)

Performing Predictive Residual Toxicity assays depends primarily on an understanding of the possible response of the stem cell population(s) in question and how the stem cell population(s) can be detected. These assays are available for lympho-hematopoietic stem cells using HALO®-96 PRT, mesenchymal stem cells using MSCGlo™-96 PRT and for other primary stem cell systems and stem cell lines using the STEMGlo™-96 PRT.  To perform a PRT assay, it may be necessary to carry out some preliminary studies.

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 (HPP-SP) and more mature hematopoietic (CFC-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 HPP-SP 1 and HPP-SP 2 primitive stem cell populations were detected. The HPP-SP 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 HPP-SP2 stem cell population has increased its sensitivity to the drug at all IC values. 

 Drug-Drug Interactions (DDI) and Combination Therapies

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.


For more information on DDI and combination therapy studies, please navigate to the DDI Page or contact HemoGenix at or call (719) 264-6250