Questions and Answers for HALO®
HemoGenix® receives many questions regarding its HALO® Platform as an alternative to and replacement for the colony-forming assay. The answers to these questions might help you decide on whether to try HALO® in your own laboratory or even dispense with the colony-forming assay entirely. If you have any questions, please contact HemoGenix® at info@hemogenix.com and we will answer them as quickly as possible and perhaps add them to this list below.
Question: Will I save time and money using HALO® Research Kit instead of the colony-forming assay?
Answer: Yes. You will save both time and money. If you presently purchase pre-mixed (but not pre-dispensed) reagents for your colony-forming assays, then you are probably purchasing bottles containing 100ml of reagents (methylcellulose, serum, growth factors etc). You use 3ml to perform 2 x 1ml cultures. This means that from a 100ml bottle of reagents, you can actually only perform 30 samples. If you were to increase the number of replicates/sample according to protocol, you would be able to perform 15 samples using 4 replicates/sample and only 10 samples at 6 replicates/sample. If you used HALO®, you would be using 100µl HALO® Master Mix/replicate. Although we do not recommend performing 2 replicates/sample for HALO®, the diagram below shows you the cost/sample of performing HALO® versus the colony-forming assay based on unit price, that is 1 bottle of 100ml colony-forming assay reagents versus a 1 x 96-well plate HALO®-96 MeC Kit.
As you see, although with increasing number of replicates the number of samples decreases, the cost rises more steeply using the colony-forming assay than with HALO®-96 MeC. In fact, even if you used just 2 replicates/sample, the cost of the colony-forming assay would increase, but that of HALO®-96 MeC would decrease, because the cost of a HALO®-96 MeC 4-plate kit or bulk order decreases considerably providing you with more savings.
The time to set up the assays is approximately the same, but the real savings comes in the time you save counting and evaluating colonies compared to measuring the luminescence in a plate luminometer. With our new 3rd generation luminescence reagents, you can now process and measure the luminescence in a full 96-well plate in 15 minutes. And this does not take into account other calculations and plotting data, which can all be performed automatically usually using the software that drives your luminometer.
Despite the need for a plate luminometer to perform HALO® and any of the other ATP-based luminescence platform, the savings in supplies, time and labor costs pay for the instrument in a very short time.
All these savings AND you get a non-subjective, fully calibrated assay.
Question: Why do I need a plate luminometer to use HALO®?
Answer: HALO® is a proliferation assay that employs a highly sensitive ATP-based, luciferin/luciferase reaction that produces bioluminescence in the form of light. The light produced in each well is actually a “glow”. You need a luminometer to detect and measure the amount of light produced. This type of light cannot be detected using an instrument that detects either absorbance or fluorescence.
Question: Will I still save money using HALO® even if I buy a plate luminometer?
Answer: Yes. The money you save in time and labor costs alone will more than pay for the cost of a plate luminometer. In fact, you would save more than $16.00/sample or between $275.00 and $300.00/96-well HALO® Research Kit, just on time and labor. If you include the cost of supplies, you would save about $350.00/HALO® kit.
Question: Which plate luminometer is right for my application?
Answer: Any plate luminometer is right for your application. There are many manufacturers. We have tested some instruments in our own laboratory. Some of these are multipramater instruments, in that they can detect absorbance, fluorescence and luminescence. They are generally cheaper to purchase and can be used for a myriad of applications. However, they are not usually as sensitive as an instrument that only measures luminescence. These “dedicated” luminometers are more expensive, but can also be obtained with up to three injectors for various applications. An extremely important aspect is the ease of use of the software that drives the instrument, not only for luminescence for all other applications the instrument has been designed for.
Question: Does HemoGenix® sell a luminometer?
Answer: HemoGenix® is working with one of the leading luminometer instrument makers to develop a dedicated machine that is very sensitive, easy to use, can be employed for multiple applications and is cost-effective to buy. HemoGenix® will be announcing this shortly.
Question: Is it necessary to use electronic pipettes?
Answer: No. But you will have greater reliability, fewer pipetting errors and therefore lower coefficients of variation if you do use electronic pipettes. It is generally good laboratory practice to have a dedicated set of pipettes for different laboratory procedures. If manual pipettes are used, they should be calibrated at regular intervals, especially if you perform procedures where you dispense microliter amounts. Any small discrepancy in pipetting can be amplified in microassays and HALO® is no exception. This can lead to outliers and statistical inaccuracies. Even though electronic pipettes are self-calibrating, they still need to be regularly checked. However, electronic pipettes are more accurate than manual pipettes and are easier to use.
Question: How do I dispense methylcellulose accurately?
Answer: Regardless of whether you are dispensing 1ml portions of methylcellulose for the colony-forming assay or only 100µl for the HALO® Master Mix, accuracy is key, and dispensing methylcellulose using a syringe and needle just does not work. The best and easiest way to dispense any methylcellulose-containing fluid accurately is to use a positive displacement repeater pipette, preferably electronic rather than manual.
Question: Can I view the cells or count clusters and/or colonies cultured in HALO®?
Answer: Yes. The 96-well plates that are included with all HALO® kits have a transparent growth surface, so you can follow the growth of the cells with time.
Question: Why does HALO® only need 7 days to detect human lympho-hematopoietic cells in culture, while the colony-forming assay requires 14 days of culture?
Answer: With HALO®, you are measuring proliferation, while with the colony-forming assay, you are measuring differentiation. At 7 days of culture, the cells are proliferating in an exponential manner, with little or no differentiation occurring (except for the CFU-E). Proliferation continues to increase exponentially until about day 10. However, after day 7, cells begin their differentiation program. After day 10, proliferation decreases and differentiation takes over. Therefore, the best time to measure proliferation of lympho-hematopoietic cells is at the point where proliferation is maximum, but little or no differentiation occurs. For the HALO®-96 MeC Platform, this time point is 7 days.
Question: Can I still detect proliferation and count colonies after 7 days in culture?
Answer: Yes. If you want to count and differentiate colonies, you can because the 96-well plates have a transparent growth surface, like the Petri dishes. You can do this at any time during culture. After counting and/or differentiating colonies, you can then process the plates and measure proliferation by luminescence. The important point to remember is that if you measure proliferation after day 7, and especially after day 10 or 12 of culture, you are actually measuring residual proliferation that is still occurring in the remaining “proliferation areas” of the colonies, while differentiation is increasing other areas of the colony.
Question: If I don’t count or distinguish colonies, how do I know that I am stimulating the right cells?
Answer: In the colony-forming assay, you add different growth factors and cytokines to stimulate different populations to differentiate into colonies. In HALO®, you are using exactly the same growth factors and/or cytokines, but you are measuring proliferation, rather than differentiation. For example, if you wanted to detect the BFU-E population in a colony-forming assay, you would stimulate the cells using either erythropoietin alone or in combination with say IL-3 and SCF. The progeny of the BFU-E would be CFU-E and other erythroblasts, which would express glycophorin-A as a membrane marker for erythroid cells. If you removed the cells from culture and performed a phenotypic analysis, you would find that the majority of the cells would actually be glycophorin-A positive. If you stimulated the BFU-E with EPO, IL-3 and SCF, you would find that, by flow cytometric analysis, not only glycophorin-A positive cells would be present, but also cells of the myelomonocytic lineage because IL-3 stimulates these cells as well as stem cells.
At HemoGenix®, we have performed extensive flow cytometric analysis for all lympho-hematopoietic cell population kits in our product line to validate our assays. The cell types you would expect to be produced as a result of stimulating specific cell populations are indeed produced. Therefore, if we know that a specific growth factor or combination of growth factors induces proliferation and steers the progeny into a specific lineage, it is not necessary to distinguish the differentiate cells morphologically because we know that these would be produced by default.
Question: How do I differentiate between the proliferation capability of cells in a lineage-specific compartment and cells that are induced from the stem cell compartment into a specific lineage?
Answer: If you were to use HALO® to detect BFU-E, you would have the choice of a kit that induced proliferation with EPO alone (designated BFU-E 1) or a kit that comes with the growth factor combination, EPO, IL-3 and SCF (designated BFU-E 2). The former measures the proliferation capability of only those cells present in the BFU-E compartment at the time point the cells are assayed. By including factors that stimulate stem cells and a factor (e.g. EPO) that can act on these cells and steer them into the erythropoietic compartment as well as induce proliferation of these progenitor cells, you are measuring a different biological process. However, by increasing the number of cells entering a specific lineage, you are also increasing the sensitivity of the assay. This could be a favorable side effect, but will depend on your study goal.
Question: Why can’t I calibrate my colony-forming assay?
Answer: Calibration refers to the process of determining the relationship between the output or response of an instrument and the values of the input quantity. This allows an assay to be standardized. You cannot calibrate the colony-forming assay because there is nothing you can calibrate the assay input (colony counts) to. There are no markers etc that allow you to perform a calibration curve to which you can compare your results. Since HALO® is based on measuring changes in intracellular ATP concentrations, the assay can be calibrated against an external ATP control to which all results can be compared. Comparing your results with results from other investigators does not calibrate or standardize the assay. It only tells you that you have produced results that are similar, but this does not allow you to directly compare your results over time on an intra- or inter-laboratory basis.
Question: How is HALO® calibrated?
Answer: HALO® is calibrated against an external ATP standard that is included with every kit. Before measuring your samples, a simple 5-point ATP dose response is performed. The output of the luminometer is Relative Luminescence Units. By performing the ATP standard dose response, all of the sample results measured in RLUs can be automatically converted to calibrated ATP concentrations (µM). The luminometer software can usually be easily programmed to perform this function and graph your results. Also included with the kit are high and low ATP controls. These controls as well as the standard curve indicate whether your instrument and reagents are working properly. They also control for pipetting errors. The small volumes dispensed means that any pipetting error is going to be amplified.
Question: How does proficiency testing help my colony-forming assay procedure?
Answer: Since the colony-forming assay is not, and cannot be standardized, proficiency testing cannot be undertaken. Furthermore, any form of proficiency testing should be controlled by an independent regulatory agency that does not stand to gain from the results. Therefore, at the present time, if you perform any type of colony-forming assay proficiency testing that is not under the auspecies of an independent agency, you should be extremely wary. Without standardization of the assay against external controls, it provides little, if any, information and no positive assurance that your procedures are correct.
Question: How can I validate my colony-forming assay?
Answer: One of the major problems of the colony-forming assay is its subjectivity in assessing and counting colonies. Unless you perform a controlled, multicenter study similar to that published by the European Center for the Validation of Alternative Methods (ECVAM), or directly compare your assay with another that can produce comparable results, the colony-forming assay cannot be validated.
Question: Has HALO® been validated?
Answer: Yes: The HALO® Platform has been validated in two ways. First, it has been validated against the colony-forming assay itself. If the total number of colonies is plotted against the ATP concentrations determined by luminescence, a direct correlation is obtained. Providing the same conditions are used for both the colony-forming assay and HALO®, then the total number of colonies produced in the manual assay can be used to obtain an ATP concentration (in µM) equivalence. The second validation of the HALO® Platform involved comparing the inhibitory concentrations at 50% (IC50 values) of at least 18 different compounds (e.g. anti-cancer drugs, anti-inflammatory drugs, chemicals etc) tested on multiple lympho-hematopoietic cell populations and comparing the results with those published in the so-called Registry of Cytotoxicity. This Registry contains the IC50 values obtained from a controlled 3T3 and human keritinocyte assay and the LD50 values obtained in mice and rats for 347 compounds. By plotting the IC50 values against the LD50 values for the same compounds tested, a linear regression should be obtained, the parameters of which must lie within certain limits for the unvalidated assayed to be considered a validated assay. HALO® conforms to this analysis and can therefore be considered a validated cytotoxicity assay. The procedure for this has been published by the National Institutes of Health as NIH Publication No.: 01-4500 and is based on recommendations by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the National Toxicology Program (NTP) and the Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM).
